U.S. patent application number 12/972315 was filed with the patent office on 2011-12-22 for composition for diagnosing parkinson's disease containing adipose tissue-derived mesenchymal stromal cell.
This patent application is currently assigned to SNU R&DB Foundation. Invention is credited to Hyo Eun Moon, Sun Ha Paek, Hyung Woo Park, Hye Young Shin.
Application Number | 20110311984 12/972315 |
Document ID | / |
Family ID | 45329006 |
Filed Date | 2011-12-22 |
United States Patent
Application |
20110311984 |
Kind Code |
A1 |
Paek; Sun Ha ; et
al. |
December 22, 2011 |
COMPOSITION FOR DIAGNOSING PARKINSON'S DISEASE CONTAINING ADIPOSE
TISSUE-DERIVED MESENCHYMAL STROMAL CELL
Abstract
The present invention relates to a composition for diagnosing a
Parkinson's disease comprising mesenchymal stromal cells derived
from adipose tissue, a method of providing information for
diagnosing Parkinson's disease and/or the extent of the disease
progression, a biomarker for diagnosing a Parkinson's disease, and
a method of screening a drug candidate treating Parkinson's disease
where the drug candidate is a target of the biomarker.
Inventors: |
Paek; Sun Ha; (Seoul,
KR) ; Moon; Hyo Eun; (Seoul, KR) ; Park; Hyung
Woo; (Incheon, KR) ; Shin; Hye Young; (Seoul,
KR) |
Assignee: |
SNU R&DB Foundation
Seoul
KR
|
Family ID: |
45329006 |
Appl. No.: |
12/972315 |
Filed: |
December 17, 2010 |
Current U.S.
Class: |
435/6.13 ;
435/325; 435/6.1 |
Current CPC
Class: |
C12N 5/0653 20130101;
G01N 2800/2835 20130101; C12N 5/0667 20130101; G01N 33/5023
20130101; C12Q 2600/136 20130101; C12Q 1/6883 20130101; C12Q
2600/158 20130101; C12N 2510/04 20130101 |
Class at
Publication: |
435/6.13 ;
435/325; 435/6.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 5/077 20100101 C12N005/077 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 18, 2010 |
KR |
10-2010-0058273 |
Claims
1. A composition for diagnosing a disease, comprising a mesenchymal
stromal cell derived from adipose tissue, wherein the disease is
selected from the group consisting of Parkinson's disease,
Alzheimer's disease, Huntington's disease, hereditary dystonia,
hereditary dyskinesia, and metabolic disease.
2. The composition according to claim 1, wherein the composition is
used for diagnosing the Parkinson's disease.
3. A method of providing information for diagnosing a disease,
comprising the steps of separating a mesenchymal stromal cell
derived from adipose tissue of a subject; assaying a gene
expression pattern of the mesenchymal stromal cell; and determining
the presence of a disease by analyzing the assayed result, wherein
the disease is selected from the group consisting of Parkinson's
disease, Alzheimer's disease, Huntington's disease, hereditary
dystonia, hereditary dyskinesia, and metabolic disease.
4. The method of providing information for diagnosing a disease
according to claim 3, wherein the disease is Parkinson's
disease.
5. The method of providing information for diagnosing a disease
according to claim 4, wherein the disease of a subject is diagnosed
to be Parkinson's disease, in case there is an increased expression
of at least one gene in the mesenchymal stromal cells derived from
the adipose tissue selected from the group consisting of
ITGA8(Genbank accession No: NM.sub.--003638), CTSH(Genbank
accession No: NM.sub.--004390), CCRL1(Genbank accession No:
NM.sub.--178445), TGFB3(Genbank accession No: NM.sub.--003239),
DRD1(Genbank accession No: NM.sub.--000794), GNA14(Genbank
accession No: NM.sub.--004297), PENK(Genbank accession No:
NM.sub.--006211), PRG4(Genbank accession No: NM.sub.--005807),
LGR5(Genbank accession No: NM.sub.--003667), HLA-DPA1(Genbank
accession No: NM.sub.--033554), SCUBE3(Genbank accession No:
NM.sub.--152753), HSPA2(Genbank accession No: NM.sub.--021979),
RELN(Genbank accession No: NM.sub.--005045), EDNRB(Genbank
accession No: NM.sub.--000115), ITGA2(Genbank accession No:
NM.sub.--002203), SLC6A6(Genbank accession No: NM.sub.--003043),
F2RL2(Genbank accession No: NM.sub.--004101), CDK6(Genbank
accession No: NM.sub.--001259), AKR1B1(Genbank accession No:
NM.sub.--001628), MMP8(Genbank accession No: NM.sub.--002424),
ID1(Genbank accession No: NM.sub.--181353), NEFM(Genbank accession
No: NM.sub.--005382), ATP1B1(Genbank accession No:
NM.sub.--001677), TNFRSF11B(Genbank accession No: NM.sub.--002546),
and TNFRSF10D(Genbank accession No: NM.sub.--003840), or a
decreased expression of at least gene selected from the group
consisting of BEX1(Genbank accession No: NM.sub.--018476),
IL8(Genbank accession No: NM.sub.--000584), and CXCL6(Genbank
accession No: NM.sub.--002993).
6. A method of screening a drug treating Parkinson's disease,
comprising the steps of: contacting a candidate compound with a
mesenchymal stromal cell derived from adipose tissue; and assaying
a gene expression pattern in the mesenchymal stromal cell, wherein
the candidate compound is determined as a drug treating Parkinson's
disease, in case difference of gene expression pattern is observed
in the mesenchymal stromal cells between the treatment and
non-treatment of the drug candidate, and the gene expression
pattern is obtained from one or more selected from the group
consisting of ITGA8(Genbank accession No: NM.sub.--003638),
CTSH(Genbank accession No: NM.sub.--004390), CCRL1(Genbank
accession No: NM.sub.--178445), TGFB3(Genbank accession No:
NM.sub.--003239), DRD1(Genbank accession No: NM.sub.--000794),
GNA14(Genbank accession No: NM.sub.--004297), PENK(Genbank
accession No: NM.sub.--006211), PRG4(Genbank accession No:
NM.sub.--005807), LGR5(Genbank accession No: NM.sub.--003667),
HLA-DPA1(Genbank accession No: NM.sub.--033554), SCUBE3(Genbank
accession No: NM.sub.--152753), HSPA2(Genbank accession No:
NM.sub.--021979), RELN(Genbank accession No: NM.sub.--005045),
EDNRB(Genbank accession No: NM.sub.--000115), ITGA2(Genbank
accession No: NM.sub.--002203), SLC6A6(Genbank accession No:
NM.sub.--003043), F2RL2(Genbank accession No: NM.sub.--004101),
CDK6(Genbank accession No: NM.sub.--001259), AKR1B1(Genbank
accession No: NM.sub.--001628), MMP8(Genbank accession No:
NM.sub.--002424), ID1(Genbank accession No: NM.sub.--181353),
NEFM(Genbank accession No: NM.sub.--005382), ATP1B1(Genbank
accession No: NM.sub.--001677), TNFRSF11B(Genbank accession No:
NM.sub.--002546), TNFRSF10D(Genbank accession No: NM.sub.--003840),
BEX1(Genbank accession No: NM.sub.--018476), IL8(Genbank accession
No: NM.sub.--000584), and CXCL6(Genbank accession No:
NM.sub.--002993).
7. The method of screening a drug treating Parkinson's disease
according to claim 6, wherein the drug candidate is determined as a
drug treating Parkinson's disease, in case the treatment of drug
candidate results in an increased expression of at least one gene
selected from the group consisting of ITGA8(Genbank accession No:
NM.sub.--003638), CTSH(Genbank accession No: NM.sub.--004390),
CCRL1(Genbank accession No: NM.sub.--178445), TGFB3(Genbank
accession No: NM.sub.--003239), DRD1(Genbank accession No:
NM.sub.--000794), GNA14(Genbank accession No: NM.sub.--004297),
PENK(Genbank accession No: NM.sub.--006211), PRG4(Genbank accession
No: NM.sub.--005807), LGR5(Genbank accession No: NM.sub.--003667),
HLA-DPA1(Genbank accession No: NM.sub.--033554), SCUBE3(Genbank
accession No: NM.sub.--152753), HSPA2(Genbank accession No:
NM.sub.--021979), RELN(Genbank accession No: NM.sub.--005045),
EDNRB(Genbank accession No: NM.sub.--000115), ITGA2(Genbank
accession No: NM.sub.--002203), SLC6A6(Genbank accession No:
NM.sub.--003043), F2RL2(Genbank accession No: NM.sub.--004101),
CDK6(Genbank accession No: NM.sub.--001259), AKR1B1(Genbank
accession No: NM.sub.--001628), MMP8(Genbank accession No:
NM.sub.--002424), ID1(Genbank accession No: NM.sub.--181353),
NEFM(Genbank accession No: NM.sub.--005382), ATP1B1(Genbank
accession No: NM.sub.--001677), TNFRSF11B(Genbank accession No:
NM.sub.--002546), and TNFRSF10D(Genbank accession No:
NM.sub.--003840), or a decreased expression of at least one gene
selected from the group consisting of BEX1(Genbank accession No:
NM.sub.--018476), IL8(Genbank accession No: NM.sub.--000584), and
CXCL6(Genbank accession No: NM.sub.--002993), relative to
non-treatment of the drug candidate.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to and the benefit of
Korean Patent Application No. 10-2010-0058273 filed in the Korean
Intellectual Property Office on Jun. 18, 2010, the entire contents
of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] (a) Field of the Invention
[0003] The present invention relates to a composition for
diagnosing a Parkinson's disease comprising mesenchymal stromal
cells derived from adipose tissue, a method of providing
information for diagnosing Parkinson's disease and/or extent of
disease progression using mesenchymal stromal cells derived from
adipose tissue, a biomarker for diagnosing a Parkinson's disease,
and a method of screening an agent treating Parkinson's disease
where the agent targets the biomarker.
[0004] (b) Description of the Related Art
[0005] Parkinson's disease (PD) is a secondarily common
neurodegenerative disease which about one percent of old people
aged over 60 suffers from, but the cause of disease has not yet
been determined.
[0006] It has been suggested that Parkinson's disease has some
connections with the selective loss of dopaminergic neurons in
substantia nigra, and with the extensive neuron changes causing
various complex motile and immotile symptoms.
[0007] A genetic mutation of disease-causing gene such as
.alpha.-synuclein, parkin, Parkinson disease autosomal recessive,
early onset 7 (DJ-1), or phosphatase and tensin homologue
(PTEN)-induced putative kinase 1 (PINK1) is mentioned as a cause of
familial Parkinson's disease.
[0008] Parkin acts as E3 ligase in ubiquitin-proteasomal system,
protects against the oxidative stress, and helps the maintenance of
mitochondrial function. The mutation in Parkin gene can cause a
hereditary early-onset of Parkinson's disease.
[0009] The correlation between mitochondrial dysfunction and
Parkinson's disease can be observed as the widely known
disease-causing mechanism in PD patient subgroup involves aberrant
shape and dysfunction of mitochondria.
[0010] The damage of mitochondrial function increases an oxidative
stress and associates with the control of calcium homeostasis and
cell apoptosis pathway. The oxidative stress can be defined as one
of causes inducing apoptosis of dopaminergic neuronal cells of the
substantia nigra in Parkinson's disease patient.
[0011] Parkinson's disease associated gene products including
.alpha.-synuclein, Parkin, PINK1, DJ-1 and the like can be found in
mitochondria and play a critical role in the mitochondrial
dysfunction and oxidative stress.
[0012] The methods of diagnosing Parkinson's disease and
determining extent of the disease progression include a method of
imaging brain nigros-triatal region with a magnetic resonance image
(MRI) analysis, a positron emission tomography (PET), a single
photon emission computed tomography (SPECT), and the like, and a
method of analyzing a sample taken from brain tissue with a
biomarker. However, such methods still lead to inaccurate analysis
of results, and unwanted pain and a risk to patient from directly
taking a sample form brain tissue.
SUMMARY OF THE INVENTION
[0013] To solve the problems in the art, the present inventors
found that Parkinson's disease could be diagnosed by using the
mesenchymal stromal cell derived from adipose tissue, developed a
technology for accurately diagnosing Parkinson's disease without
directly taking brain tissue, and completed the present invention
by developing a biomarker for diagnosing Parkinson's disease using
the technology.
[0014] Therefore, an embodiment of the present invention provides a
composition for diagnosing Parkinson's disease comprising the
mesenchymal stromal cell derived from adipose tissue.
[0015] Another embodiment provides a method of providing an
information for diagnosing Parkinson's disease and determining
extent of the progression of Parkinson's disease.
[0016] Further embodiment of the present invention provides a
biomarker for diagnosing Parkinson's disease where the biomarker is
obtained from the mesenchymal stromal cell derived from the adipose
tissue.
[0017] Still another embodiment of the present invention provides a
method of screening a drug treating Parkinson's disease where the
drug targets the biomarker.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0018] The present invention can provide a tool for understanding
and determining the brain physiological states without isolating
the brain tissue from Parkinson's disease patient, by performing
the transcriptome microarray analysis of mesenchymal stromal cell
derived from early-passage adipose tissue taken from human subject
with early-onset (congenital) hereditary Parkinson's disease such
as Parkin-deficient PD, as well as late-onset (acquired) Idiopathic
Parkinson's disease (idiopathic PD).
[0019] The present inventors completed the present invention by
separating a human mesenchymal stromal cell derived from human
adipose tissue (hAD-MSC), from a subject with Idiopathic PD or
Parkin-deficient Parkinson's disease, and comparing the gene
expression pattern of the mesenchymal stromal cell with that of
non-Parkinson's disease. The human adipose tissue is abundant and
easily accessible source for mesenchymal stem cells (MSC).
[0020] Hereinafter, hAD-MSC obtained from a patient with idiopathic
Parkinson's disease is referred to as `PD,` hAD-MSC obtained from a
patient with Parkin-deficient Parkinson's disease as `Parkin,` and
hAD-MSC obtained from a patient who has pituitary adenoma without
Parkinson's disease as `non-PD` or `PA.` Initially, by analyzing
differentially-expressed gene (DEG) among three groups, 413 genes
are confirmed to be differentially expressed, and then classified
into three groups of non-PD, PD and Parkin. In addition, DEG are
analyzed and divided to seven clusters according to K-mean
clustering analysis, and the genbank accession numbers are listed
in Tables 6a-6e. In addition, the functional groups of human
biomarker candidates are organized, and non-PD vs. PD and non-PD
vs. Parkin are compared. Finally, the PD associated DEGs which are
regulated differently due to the oxidative stress are classified
into one of groups among non-PD, PD and Parkin categories.
[0021] The knowledge of selective gene expression pattern in
Parkinson's disease patient gained from the present invention can
be helpful for obtaining the physiological information and
early-diagnosis of Parkinson's disease, and developing an effective
target specific drug for treating the Parkinson's disease by using
the genes as a biomarker.
[0022] First of all, the present invention relates to a composition
for diagnosing a disease, comprising mesenchymal stromal cells
derived from adipose tissue, where the disease is selected from the
group consisting of Parkinson's disease, Alzheimer's disease,
Huntington's disease, hereditary dystonia, hereditary dyskinesia,
and metabolic disease. The present inventors found that the
presence of Parkinson's disease and the extent of disease
progression could be diagnosed without separating the brain tissue
from Parkinson's disease patient by assaying the gene expression
pattern of mesenchymal stromal cell derived from adipose tissue,
since the gene expression is very similar to that of brain tissue.
In accordance with the present invention, the use of adipose
tissue-derived mesenchymal stromal cell for diagnosing the
Parkinson's disease removes the step of dangerous separation of
brain tissue from the subject. The diagnosing method using the
adipose tissue-derived mesenchymal stromal cells can be widely
applied for diagnosing various diseases such as Alzheimer's
disease, Huntington's disease, hereditary dystonia, hereditary
dyskinesia, and metabolic disease.
[0023] The subject includes any kind of mammals, and preferably
human beings who are suffering or are likely to suffer from
Parkinson's disease, Alzheimer's disease, Huntington's disease,
hereditary dystonia, hereditary dyskinesia, and metabolic disease.
Since one of the common causes of the diseases is adipose tissue
(Human Lipodystrophies: Genetic and Acquired Diseases of Adipose
Tissue and http://www.ncbi.nlm.nih.gov/pubmed/20551664), the
application of following diagnosing technology for Parkinson's
disease can be extended to diseases listed above.
[0024] The adipose tissue can be one separated from mammals,
preferably human. The separated region is not limited to a
particular region of body and can include any regions of a body
such as breast and abdominal region. For more accurate analysis,
the separated adipose tissue can be used preferably after
eliminating cell debris and blood cell.
[0025] Hereinafter, the term, adipose tissue can be separated or
unseparated one from a live body, and includes an adipose cell.
[0026] In an embodiment of the present invention, the mesenchymal
stromal cells (hAD-MSC) can be, for example, but are not limited
thereto, cells showing the mononuclear cell properties which are
confirmed by positive expression of human integrin beta-1 marker
CD29, phagocytic glycoprotein-1 marker CD44, and human integrin
alpha-4 marker CD49d, and at the same time expressing slightly
primitive hematopoietic precursors and vascular endothelial marker
CD34, vascular endothelial marker CD31 and vascular adhesion
molecule 1, (VCAM-1) marker CD106.
[0027] The Parkinson's diseases which can be detected by using the
composition of the present invention includes all kinds of the
Parkinson's diseases such as an acquired Idiopathic Parkinson's
disease, congenital, familial Parkinson's disease (for examples,
Parkin, .alpha.-synuclein, phosphatase and tensin homologue
(PTEN)-induced putative kinase 1 (PINK1, a mitochondrial kinase),
Parkinson disease autosomal recessive, early onset 7 (DJ-1),
Leucine-rich repeat kinase 2 (LRRK2), and High temperature
requirement protein A2 (HTRA2) deficient).
[0028] In another embodiment of the present invention, a method of
diagnosing Parkinson's disease using the mesenchymal stromal cells
derived from the adipose tissue is provided.
[0029] More specifically, the method comprises the steps of
separating a mesenchymal stromal cell derived from adipose tissue
of a subject, assaying a gene expression pattern of the mesenchymal
stromal cell; and determining the presence of disease by analyzing
the assayed result or the disease progression degree by analyzing
the assayed result.
[0030] The assaying step of gene expression pattern of hAD-MSC can
be performed by any methods of analyzing gene expression which are
used commonly in the art. For examples, the assaying of gene
expression can be carried out by microarray analysis, Reverse
transcriptase Polymerase Chain Reaction (RT-PCR), Real time
Polymerase Chain Reaction (real time PCR), genomics, proteomics,
microRNA assay, SNP analysis, mitochondrial assay, functional assay
and the like, but not limited thereto.
[0031] As a result of the molecular biological studies in the
present invention, a high-throughput microarray analysis of hAD-MSC
obtained from a patient with Idiopathic Parkinson's disease, a
patient with Parkin-deficient Parkinson's disease, and a patient
with non-Parkinson's disease can be established, and the analysis
result is compared with that of a patient with non-Parkinson's
disease (control group) to identify the gene groups which are
differentially expressed between a patient with Idiopathic
Parkinson's disease and/or a patient with Parkin-deficient
Parkinson's disease. Thus, the identified genes in a patient with
non-Parkinson's disease, a patient with Idiopathic Parkinson's
disease and a patient with Parkin-deficient Parkinson's disease can
contribute to the understanding of physiological symptoms of
Parkinson's disease, and can provide a useful tool for developing
the early-diagnosis and effective treatment of Parkinson's disease
targeting the human biomarker.
[0032] The mitochondrial dysfunction and increased oxygen stress
are shown in subgroup of Parkinson's disease patient, and these
suggest the important effect of mitochondrial dysfunction and
oxygen stress on onset of Parkinson's disease. Thus, the
mitochondria can be effective target for developing a biomarker of
Parkinson's disease. The biochemical methods of detecting the
potential biomarker of Parkinson's disease include the gene
screening method, mitochondria complex I measurement, blood level
of alpha-synuclein and isoforms measurement. Gene test tools which
are commercially available for detecting mitochondria mutant genes
such as Parkin, PINK1, and alpha-synuclein can be used.
Furthermore, Coenzyme Q10, antioxidant and electron transporter
(electron transporter chain component) act as an electron
transporter for mitochondria complex I and II.
[0033] The genes which show differential expression at least two
times in non-PD patient vs. Parkin, and non-PD vs. PD, and Parkin
vs. PD are identified with hierarchical clustering analysis (FIG.
4), and summarized in table 4 to select the PD-related genes
(SCUBE3, IL8, ATP1B1, TNFRSF11B, FABP3, CXCL1). IL8 and CXCL1 are
chemokines which act a basic role in development, homeostasis and
immune systems, and involve in inflammation of cranial nerve of
Parkinson's disease patient. SCUBE3 accompanies an important
molecule in dopaminergic neuron of ventral midbrain, and TNFRSF11B
involves in an inflammation in neurodegeneration of Parkinson's
disease. The single heterozygous mutation of ATP1B1 is suggested to
be related with a cause of early-onset of Parkinson's disease.
Finally, FABP3 has been used as a diagnostic marker for Parkinson's
disease. The PD-related genes in non-PD vs. Parkin and Parkin vs.
PD are summarized in Table 5 (SYT14, LGR5, TGFB3, ITGA2, F2RL2,
DRD1, PENK, GNA14, EDNRB, HSPA2, SLC6A6, AKR1B1, and PRG4). SYT14
is a transmembrane protein involved with control of membrane
trafficking. LGR5, F2RL2, DRD1, GNA14 and EDNRB involve in a signal
pathway of G-protein, TGFB3 involves in a susceptibility of
Parkinson's disease patient, and ITGA2 in a neuronal adhesion. An
increased expression of PENK can be a cause of treatment-related
dyskinesia in Parkinson's disease patient. Parkin, as a substrate
for parkin, mediates the ubiquitination of HSPA2 and a molecular
chaperone. SLC6A6, also known as taurine, is a neurotransmitter.
Taurine is a beta-amino acid abundantly located in substantia nigra
(SN), and functions as a neurotransmitter in substantia. The
immunoreactivity of AKR1B1 is generated in human cerebral cortex
and hippocampus, and PRG4 relates with the inclusions of
Parkinson's disease.
[0034] The comparison analysis of seven regulating sequences of
K-mean clustering genes in non-PD, PD and Parkin are shown in FIG.
5b. The names and genbank accession numbers of PD-related genes
showing the increased or decreased gene expression are summarized
in Tables 6a-6e. The genes showing the increased gene expression
pattern are shown in Table 6a (ITGA8, CTSH, CCRL1), Table 6b
(TGFB3, DRD1, GNA14, PENK, PRG4, LGR5, HLA-DPA1), and Table 6e
(SCUBE3, HSPA2, TGFB3, DRD1, GNA14, PENK, PRG4, LGR5, RELN, EDNRB,
ITGA2, SLC6A6, F2RL2, CDK6, AKR1B1, MMP8, ID1, NEFM, ATP1B1,
TNFRSF11B, TNFRSF10D), and the genes showing the decreased gene
expression pattern are shown in Table 6c (BEX1) and Table 6d (IL8,
CXCL6). The change in gene expression seems to be due to the
mitochondria activity change between late-onset of Idiopathic
Parkinson's disease and congenital early-onset of Parkinson's
disease or due to compensation therebetween. These data also
suggest the potential biomarker for the onset of Parkinson's
disease. The molecular functional groups in non-PD vs. PD and
non-PD vs. Parkin are determined and shown in FIG. 8a to FIG. 8d,
and the genes which show the change in the gene expression level in
Idiopathic Parkinson's disease patient and Parkin-deficient
Parkinson's disease patient can provide a potential human biomarker
candidate for detecting a disease onset and a selective
vulnerability.
[0035] In addition, after analyzing of genes differentially
expressed due to the oxygen stress in PD, the genes and the
clusters taken from Cluster Nos. 2 to 6 are shown in Table 9.
Interestingly, genes showing a linear increase of gene expression
were discovered in all groups, which may be explained by an
increased compensation against vulnerability caused by oxygen
stress in pathology of Idiopathic PD and Parkin-deficient PD.
[0036] Based on these results, when there is an increased
expression of at least one selected from the group consisting of
ITGA8, CTSH, CCRL1, TGFB3, DRD1, GNA14, PENK, PRG4, LGR5, HLA-DPA1,
SCUBE3, HSPA2, RELN, EDNRB, ITGA2, SLC6A6, F2RL2, CDK6, AKR1B1,
MMP8, ID1, NEFM, ATP1B1, TNFRSF11B, and TNFRSF10D in mesenchymal
stromal cells derived from adipose cell of a patent, or when where
is an decreased expression of at least one selected from the group
consisting of BEX1, IL8, and CXCL6 in mesenchymal stromal cells
derived from adipose cell of a patent, it is possible to determine
the presence of the Parkinson's Disease. Parkinson's disease
includes late-onset (acquired) Parkinson's disease (Idiopathic
Parkinson's disease), or early-onset (congenital, familial,
hereditary) Parkinson's disease (for examples, Parkin,
.alpha.-synuclein, phosphatase and tensin homologue (PTEN)-induced
putative kinase 1 (PINK1, a mitochondrial kinase), Parkinson
disease autosomal recessive, early onset 7 (DJ-1), Leucine-rich
repeat kinase 2 (LRRK2), or High temperature requirement protein A2
(HTRA2) deficient Parkinson's disease and etc).
[0037] In an embodiment of the present invention, the increase and
decrease of the gene expressions can be measured by the amount of
protein expressed. When the amount is about 1.5 to 3 times higher
than that of normal group without Parkinson's disease, the result
can be determined to be significant.
[0038] In an embodiment of the present invention, a method of
screening a drug treating Parkinson's disease, in which the drug
targets the biomarker in mesenchymal stromal cells derived from
adipose tissue, is provided.
[0039] More specifically, the method comprises the steps of
contacting a drug candidate with a mesenchymal stromal cell derived
from adipose tissue; and assaying a gene expression pattern of the
mesenchymal stromal cell; and determining the drug candidate as a
drug treating Parkinson's disease in case that there is a
difference of gene expression pattern in the mesenchymal stromal
cells between the treatment and non-treatment of the drug
candidate, wherein at least gene is selected from the group
consisting of ITGA8, CTSH, CCRL1, TGFB3, DRD1, GNA14, PENK, PRG4,
LGR5, HLA-DPA1, SCUBE3, HSPA2, RELN, EDNRB, ITGA2, SLC6A6, F2RL2,
CDK6, AKR1B1, MMP8, ID1, NEFM, ATP1B1, TNFRSF11B, TNFRSF10D, BEX1,
IL8, and CXCL6.
[0040] For example, when the group treated with the drug candidate
shows the increased gene expression (preferably, at least an
increase of two-fold) of at least one selected from the group
consisting of ITGA8, CTSH, CCRL1, TGFB3, DRD1, GNA14, PENK, PRG4,
LGR5, HLA-DPA1, SCUBE3, HSPA2, RELN, EDNRB, ITGA2, SLC6A6, F2RL2,
CDK6, AKR1B1, MMP8, ID1, NEFM, ATP1B1, TNFRSF11B, TNFRSF10D and the
like, or the decreased gene expression (preferably, at least a
decrease of two-fold) of at least one selected from the group
consisting of BEX1, IL8, CXCL6 and the like, compared to that of
the group untreated with the drug candidate, the drug candidate can
be determined as a drug for treating Parkinson's disease, for
examples, late-onset (acquired) Parkinson's Disease (Idiopathic
Parkinson's Disease), or early-onset (congenital, familial,
hereditary) Parkinson's Disease (for examples, Parkin,
.alpha.-synuclein, phosphatase and tensin homologue (PTEN)-induced
putative kinase 1 (PINK1, a mitochondrial kinase), Parkinson
disease autosomal recessive, early onset 7 (DJ-1), Leucine-rich
repeat kinase 2 (LRRK2), or High temperature requirement protein A2
(HTRA2) deficient Parkinson's disease, etc.
[0041] The increase or decrease of gene expression can be measured
by any method of measuring the gene expression level which has been
known generally in the art, for examples but not limited to,
microarray assay, Reverse transcriptase polymerase chain reaction
(RT-PCR), Real time PCR, genomics, proteomics, microRNA assay, SNP
analysis, mitochondrial assay, functional assay and the like.
[0042] The data obtained in the present invention provide a
predictable scenario for onset of Parkinson's disease. In
conclusion, the gene expression analysis of the mesenchymal stromal
cells derived from the human adipose tissue can identify specific
molecular functional groups of the genes which are affected by the
mitochondrial dysfunction and oxidative stress. Thus, the present
invention provides a technology for diagnosing Parkinson's disease
and/or determining its extent of disease progression using the
mesenchymal stromal cell derived from adipose tissue instead of
brain tissue, for. The genes which show the change in gene
expression in Idiopathic or Parkin-deficient (familial) Parkinson's
disease patient compared to the non-Parkinson's disease can be
identified by using such technology, and can be used both as a
biomarker as well as a target for developing a drug treating
Parkinson's disease.
BRIEF DESCRIPTION OF DRAWINGS
[0043] FIGS. 1a and 1b show the procedure of separating and
culturing the mesenchymal stromal cells derived from adipose tissue
of human patient with Parkinson's disease.
[0044] FIG. 2 represents schematically the procedure of preparing a
stock by culturing the mesenchymal stromal cells derived from
adipose tissue of human patient with Parkinson's disease.
[0045] FIG. 3 is a picture showing the transient change in cell
morphology of the mesenchymal stromal cells derived from adipose
tissue of human patient with Parkinson's disease based on culture
time variations.
[0046] FIG. 4 is a Venn diagram of Differentially Expressed Gene
(DEG) between the control group (non-PD, PA) and test group with
Parkinson's disease.
[0047] FIG. 5a is a result of hierarchical clustering analysis of
the genes showing the gene expression level at least two times
higher between non-PD, PD and Parkin groups.
[0048] FIG. 5b shows the seven clusters obtained by reorganizing
the expression pattern graphs which are classified through the
K-mean clustering analysis of non-PD, PD and Parkin.
[0049] FIG. 6 represents the clustering analysis result of the
genes showing linearly-decreased gene expression between non-PD,
PD, and Parkin groups.
[0050] FIG. 7 represents the clustering analysis result of the
genes showing linearly-increased gene expression between non-PD,
PD, and Parkin groups.
[0051] FIG. 8a to 8d show the result classifying human biomarker
which are obtained by reprogramming the molecular functional groups
by using Gene Ontology and Panther database system between non-PD
vs. PD and non-PD vs. Parkin.
[0052] FIG. 9 shows the comparison of cell morphology before and
after immortalization with hTERT in non-PD, PD, and Parkin
group.
[0053] After: the cell morphology shortly after immortalization
with hTERT.
[0054] 6 months: the cell morphology after immortalization with
hTERT and culturing for 6 months.
[0055] 1 year: the cell morphology after immortalization with hTERT
and culturing for 1 year.
[0056] FIG. 10 shows a result of chromosomal structure (karyotype)
analysis.
[0057] after: the karyotype analysis result shortly after
immortalization with hTERT.
[0058] 1 year: the karyotype analysis result after immortalization
with hTERT and culturing for 1 year.
[0059] FIG. 11a shows a result of biochemical enzyme assay of
mitochondria respiration chain of immortalized cell, and FIG. 11b
is the result of western blot analysis showing successful
separation of mitochondria in the assay of FIG. 11a.
[0060] FIG. 12 is electronic microscopic images of mitochondria in
early-culture stage and immortalization-culture stage of the cells
(a, b: mitochondria in Non-PD; c, d: mitochondria in PD; and e, f:
mitochondria in Parkin).
[0061] FIG. 13a represents a result of western blot analysis (left
side) which shows the change in the gene expression of
mitochondrial markers in early-culture stage of non-PD, PD, and
Parkin groups, and a graph (right side) showing the quantitative
analysis of the result.
[0062] FIG. 13b represents the change in the gene expression of
HSP60 as mitochondria marker in immortalization-culture stage of
the cells in non-PD, PD, and Parkin (left side) and a graph (right
side) showing the quantitative analysis of the result.
[0063] FIG. 13c represents the change in the gene expression of
HSP90 as mitochondria marker in immortalization-culture stage of
the cells in non-PD, PD, and Parkin (left side) and a graph (right
side) showing the quantitative analysis of the result.
[0064] FIG. 14a represents a western blot analysis showing the
change in the gene expression of autophagy makers in
immortalization-culture stage of the cells in non-PD, PD, and
Parkin.
[0065] FIG. 14b represents a western blot analysis showing the
change in the gene expression of mTOR as an autophagy maker in
immortalization-culture stage of the cells in non-PD, PD, and
Parkin of FIG. 14a.
[0066] FIG. 14c represents a western blot analysis showing the
change in the gene expression of S6K as an autophagy maker in
immortalization-culture stage of the cells in non-PD, PD, and
Parkin of FIG. 14a.
[0067] FIG. 15a represents a western blot analysis showing the
change in the gene expression of autophagy markers in
immortalization-culture stage of the cells in non-PD, PD, and
Parkin, and FIG. 15b is a graph showing the quantitative analysis
of the result.
[0068] FIG. 16a represents western blot analysis showing the change
in the gene expression of autophagy markers in
immortalization-culture stage of the cells in non-PD, PD, and
Parkin.
[0069] FIG. 16b is a graph showing the quantitative analysis of the
change in the gene expression of autophagy markers in
immortalization-culture stage of the cells in non-PD, PD, and
Parkin.
[0070] FIG. 17 shows an analysis result of properties of the
mesenchymal stromal cells derived from the adipose tissue.
EXAMPLE
[0071] The present invention is further explained in more detail
with reference to the following examples. These examples, however,
should not be interpreted as limiting the scope of the present
invention in any manner.
Example 1
Preparation of the Mesenchymal Stromal Cell Derived from the
Adipose Tissue
[0072] 1.1. Separation and Culture of the Mesenchymal Stromal Cells
Derived from the Adipose Tissue
[0073] The adipose tissue-derived mesenchymal stromal cells
(hAD-MSC) were separated from Idiopathic Parkinson's disease
(Idiopathic PD) patient, Parkin-deficient Parkinson's disease
(Parkin-deficient PD) patient, and a pituitary adenoma patient who
did not suffer from Parkinson's disease, and then subsequently were
cultured. Hereinafter, otherwise particularly defined, hAD-MSC
obtained from Idiopathic PD patient is referred to as "PD," hAD-MSC
obtained from Parkin-deficient Parkinson's disease patient to as
"Parkin", and hAD-MSC obtained from Parkinson's disease to as
"non-PD" or "PA."
[0074] These tests were performed under the permission of
Institutional Review Board of Seoul National University Hospital
(IRB No. 0707-024-212) and the written consent of the patients.
During the performance of Deep Brain Stimulation (DBS) surgery of
Idiopathic PD patient, and Parkin-deficient PD patient, the adipose
tissue under the skin of clavicle was taken in order to compare
with that of a pituitary adenoma patient (control group) who did
not suffer from Parkinson's disease.
[0075] The adipose tissue was added to 1% antibiotic/antimyotic
(Gibco.RTM.Invitrogen, Carlsbad, Calif.) in sterilized PBS
(phosphate-buffered saline, pH7.4) and transferred to test room.
The adipose tissue was washed with PBS three times to remove tissue
debris and red blood cell, and then finely cut into small pieces.
The adipose tissue was digested with 0.075% collagenase Type I
(Sigma-Aldrich, St. Louis, Mo., USA) at 37.degree. C. for 1 hour,
inactivated with the same volume of DMEM/10% Fetal Bovine Serum
(FBS) (Gibco.RTM. Invitrogen, Carlsbad, Calif.), and centrifuged at
1200.times.g for 10 minutes. The obtained pellet was cultured in
three times volume of red blood lysis buffer (QIAGEN, valencia, CA,
USA) at 37.degree. C. for 10 minutes, and filtered with 100 .mu.M
strainer. The filtrate was centrifuged at 1200.times.g for 10
minutes. The resultant pellet was washed with PBS and centrifuged
at 1200.times.g for 10 minutes. Finally, the obtained pellet was
resuspended in Mesenchymal Stem cell Expansion medium (Millipore,
SCM015, Billerica, Mass., USA) and placed onto 25T culture flask.
After the cells were cultured in Mesenchymal Stem cell Expansion
medium (Millipore, SCM015, Billerica, Mass., USA) at 37.degree. C.
for 48 hours, the cells were washed with PBS and the unattached
cells were removed. The culture medium was replaced with new medium
every three days.
[0076] As a result, the hAD-MSC was obtained. Among the hAD-MSC
obtained, two cells obtained from a pituitary adenoma patient who
did not suffer from Parkinson's Disease (PA1 and PA2), two cells
obtained from Idiopathic PD (PD1 and PD2), and two cells obtained
from Parkin-deficient PD (Parkin) and Parking) were deposited at
Korean cell line bank located at 28 Yongon-dong, Chongno-gu, Seoul
110-744, Korea on Nov. 17, 2010, and then assigned with the
accession numbers of KCLRF-BP-00239(PA1), KCLRF-BP-00240(PA2),
KCLRF-BP-00241(PD1), KCLRF-BP-00242(PD2), KCLRF-BP-00243(Pakin1),
and KCLRF-BP-00244(Pakin2).
[0077] The culture procedure is shown schematically in FIGS. 1a and
1b. FIG. 2 represents schematically the procedure of preparing
stock by culturing mesenchymal stromal cells derived from adipose
tissue of human patient with Parkinson's disease. FIG. 3 is a
picture showing the transient change in cell morphology of
mesenchymal stromal cells derived from adipose tissue of human
patient with Parkinson's disease according to the culture time
variations. In FIG. 3, the pictures of cell morphology of adipose
tissue-derived mesenchymal stromal cells were taken every day
during continuous culture.
[0078] The information on the cell culture of mesenchymal stromal
cells derived from adipose tissue obtained from Idiopathic PD
patient, Parkin-deficient PD patient, and control group are
summarized at Tables 1 to 3:
TABLE-US-00001 TABLE 1 Culture information of adipose tissue
obtained from Idiopathic PD patient Patient No. Labeling Culture
Date 1 FSC-PD#2 2007-03-09 2 FSC-PD#3 2007-04-02 3 FSC-PD#5
2007-08-13 4 FSC-PD#6 2007-10-22 5 FSC-PD#7 2007-10-29 6 FSC-PD#8
2007-11-19 7 FSC-PD#9 2008-03-24 8 FSC-PD#10 2008-07-07 9 FSC-PD#11
2008-08-29 10 FSC0714 2008-07-14 11 FSC0721 2008-07-21 12 FSC0829
2008-08-29 13 FSC1006 2008-10-06 14 FSC0119 2009-01-19 15 FSC0209
2009-02-09 16 FSC0420 2009-04-20 17 FSC0427 2009-04-27 18 FSC0601
2009-06-01 19 FSC0622 2009-06-22 20 FSC0918 2009-09-18 21 FSC1123
2009-11-23
TABLE-US-00002 TABLE 2 Culture information of adipose tissue
obtained from Parkin-deficient PD patient Patient Labeling Culture
Date 1 FSC-parkin 2007-05-17 2 gFSC 2008-06-02
TABLE-US-00003 TABLE 3 Culture information of adipose tissue
obtained from control group Patient No. Labeling Culture Date 1
FSC-#1 2006-11-22 2 FSC-#2 2006-11-23 3 FSC-#3 2006-12-18 4 FSC-#4
2006-12-18 5 FSC-#7 2007-01-08 6 FSC-#8 2007-01-22 7 FSC-#9
2007-01-31 8 FSC-#11 2007-02-15 9 FSC-#12 2007-02-26 10 FSC-#14
2007-03-15 11 FSC-#15 2007-04-20 12 FSC-#17 2007-10-02 13 FSC-#18
2007-10-04 14 FSC-#19 2008-03-25 15 FSC1013 2008-10-13 16 FSC1014
2008-10-14 17 FSC1103 2008-11-03 18 FSC1104 2008-11-04 19 FSC0629
2009-06-29 20 FSC0630 2009-06-30 21 FSC0706 2009-07-06 22 FSC1019
2009-10-19 23 FSC1102 2009-11-02 24 FSC1109 2009-11-09 25 FSC1201
2009-12-01
[0079] 1.2. Fluorescence-Activated Cell Sorter (FACS) Analysis
[0080] The hAD-MSC culture was separated with PBS and subsequently
cultured with following primary antibodies (culture medium:
Mesenchymal Stem cell Expansion medium (Millipore, SCM015,
Billerica, Mass., USA), culture temperature: 37.degree. C.).
[0081] Primary Antibody:
[0082] anti-CD29, anti-CD44, anti-CD34, anti-CD31 (DakoCytomation,
Carpinteria, Calif., USA), anti- and CD49d, anti-CD106 (Chemicon,
Temecula, Calif., USA).
[0083] The cells were cultured on ice for 30 minutes, and then
washed with 0.5% BSA and 2 mM EDTA in BSA (Sigma-Aldrich, St.
Louis, Mo., USA). The morphological characteristics of hAD-MSC and
quantitative analysis were performed by FACS SCAN flow cytometer
(Becton Dickinson, San Diego, Calif., USA) and CellQuest software
(Becton Dickinson, San Diego, Calif., USA).
[0084] The results are shown in FIG. 17. hAD-MSC obtained from
idiopathic PD patient, Parkin-deficient PD patient, and control
group were separated and cultured. Then, the cells showed the
characteristics of mononuclear cell based on the expression of
human integrin beta-1 marker CD29, phagocytic glycoprotein-1 marker
CD44, and human integrin alpha-4 marker CD49d. In addition, the
cells expressed slightly primitive hematopoietic precursors and
vascular endothelial marker CD34, vascular endothelial marker CD31
and vascular adhesion molecule 1, (VCAM-1) marker CD106.
[0085] 1.3. Preparation of RNA Sample
[0086] According to manufacturer's manual, the RNA sample was
prepared. Specifically, whole RNA was separated with RNeasy Mini
Kit columns (Qiagen, Hilden, Germany) according to the
manufacturer's manual. The quantity of RNA was assessed with
Agilent 2100 bioanalyser using RNA 6000 Nano Chip (Agilent
Technologies, Amstelveen, The Netherlands) and determined with
ND-1000 Spectrophotometer (Nanoprop Technologies, Inc., DE,
USA).
[0087] 1.4. Analysis of hAD-MSC Properties
[0088] hAD-MSC was separated from Idiopathic PD patient,
Parkin-deficient PD patient, and the control group and cultured. In
flow cytometry analysis, at least 95% of MSC (.gtoreq.95%)
expressed CD105, CD73 and CD90, and the cells were deficient in the
expression of CD45, CD34, CD14 or CD11b; CD79.quadrature. or CD19;
and HLA class II (positive at most 2%) (M Dominici, K Le Blanc, I
Mueller, I Slaper-Cortenbach, F Marini, D Krause, et al, Minimal
criteria for defining multipotent mesenchymal stromal cells. The
International Society for Cellular Therapy position statement,
Cytotherapy, 8, 315-7, 2006). The accession numbers of the genes
are summarized in following table:
TABLE-US-00004 Gene name Gene accession number CD105 NM_001114753
CD73 NM_002526 CD90 NM_006288 NM_033209 CD45 NM_002838 CD34
NM_001025109 CD14 NM_000591 CD11b NM_000632 CD79 NM_001783 CD19
NM_001178098 HLA class II NM_000449
[0089] The cells should be able to differentiate into osteoblasts
cell, adipose cell (adipocytes) and chondroblasts under the
standard in vitro differentiation condition (M Dominici, K Le
Blanc, I Mueller, I Slaper-Cortenbach, F Marini, D Krause, et al,
Minimal criteria for defining multipotent mesenchymal stromal
cells. The International Society for Cellular Therapy position
statement, Cytotherapy, 8, 315-7, 2006).
[0090] The separated hAD-MSC were cultured at Mesenchymal Stem cell
Expansion medium (Millipore, SCM015, Billerica, Mass., USA) at
37.degree. C., and the expression profile of human integrin beta-1
marker CD29, phagocytic glycoprotein-1 marker CD44, and human
integrin alpha-4 marker CD49d were analyzed with FACS analysis
method. The result is shown in FIG. 17. CON in FIG. 17 shows FACS
analysis result of mesenchymal stromal cells themselves.
[0091] The accession numbers of the genes utilized are summarized
in following table:
TABLE-US-00005 Gene name Gene accession number CD29 NM_002211 CD44
NM_000610 CD49d NM_000885 CD34 NM_001025109 CD31 NM_000558 CD106
NM_001078
[0092] The positive expression of the genes confirmed that the
cells showed the properties of the mononuclear cell. The cells
expressed a small amount of primitive hematopoietic precursors and
vascular endothelial marker CD34, vascular endothelial marker CD31
and vascular adhesion molecule 1, (VCAM-1) marker CD106.
Example 2
Gene Profiling of Adipose Tissue-Derived Mesenchymal Stromal
Cell
[0093] 2.1. cDNA Microarray Analysis
[0094] The gene expression analysis was performed with Affymetrix
GeneChip.RTM. Human Gene 1.0 ST oligonucleotide array (DNA LINK,
INC (Seoul, Korea)).
[0095] According to Affymetrix manufactor's protocol
(http://www.affymetrix.com), 300 ng of RNA was added to each
sample. Namely, 300 ng of all RNA per a sample was changed into
double stranded cDNA. The double stranded cDNA was obtained by
using SuperScrpit II Reverse Transcriptase, DNA polymerase I and
random hexamer inserted by T7 promoter (Affymetrix GeneChip.RTM. WT
cDNA Synthesis and Amplipicaition Kit, Cat No. 900672). An
amplified RNA (cDNA) was produced by in vitro transcription (IVT)
with IVT Enzyme Mix (Affymetrix GeneChip.RTM. WT cDNA Synthesis and
Amplipicaition Kit, Cat No. 900672), and separated with Affymetrix
sample cleanup module. The amplified cRNA was mixed with IVT cRNA
binding buffer and 100% EtOH, and bonded in cRNA cleanup spin
column. The column was washed with cRNA wash buffer, and then
eluted with RNase-free water.
[0096] cDNA was reproduced with dNTP mixture including dUTP
Affymetrix GeneChip.RTM. WT cDNA Synthesis and Amplipicaition Kit,
Cat No. 900672), according to the random-primed reverse
transcription. Then, the produced cDNA was fragmented by using UDG
and APE 1 restriction enzyme (Affymetrix GeneChip.RTM. WT Terminal
Labeling Kit, Cat No. 900670), and the end labeling was performed
by inserting biotinylated dideoxynucleotide with TdT (Terminal
deoxynucleotidyl transferase) enzyme.
[0097] According to Gene Chip Whole Transcript (WT) Sense Target
Labeling Assay Manual (Affymetrix), the end-labeled and fragmented
cDNA was hybridized at 60 r/min, at 45.degree. C., for 16 hours
with GeneChip.RTM. Human Gene 1.0 ST array. Then, the array was
stained and washed in Genechip Fluidics Station 450 (Affymetrix),
and scanned with Genechip Array scanner 3000 7G (Affymetrix).
[0098] 2.2. Classification of DEG Included in Three Groups of
Non-PD (Control Group), Idiopathic PD and Parkin-Deficient PD
[0099] To identify DEG based on two-fold gene expression difference
between non-PD vs. Parkin, non-PD vs. PD and Parkin vs. PD,
hierarchical clustering analysis (Eisen M B, Spellman P T, Brown P
O, Botstein D, 1998) Cluster analysis and display of genome-wide
expression patterns. Genetics Vol. 95, Issue 25, 14863-14868) was
performed. The hierarchical clustering analysis is data-mining
algorithm used for defining similarity or dissimilarity of
expressed genes. By using all genes showing the gene expression
difference of two-fold between three groups of non-PD, PD, Parkin
as a standard, the hierarchical clustering analysis was carried out
to identify the gene having high similarity and the Euclidean
distance was used as a similarity measurement.
[0100] The obtain result is shown as a Venn diagram in FIG. 4. In
order to identify genes showing expression level difference of at
least two-fold between the groups of non-PD, PD and Parkin, after
selecting genes showing expression level difference of at least
two-fold in each non-PD vs Parkin, non-PD vs PD, and Parkin vs PD,
and comparing with the results among non-PD vs Parkin, non-PD vs
PD, and Parkin vs PD, the genes that showed the similar level of
difference in each comparison were selected and shown in FIG.
4.
[0101] For example, the genes having gene expression level of at
least two times higher between the groups of non-PD vs. Parkin were
109 genes which included 20 genes showing two-fold expression in
comparison of non-PD vs. Parkin and 16 genes showing two-fold
expression level in all three comparisons.
[0102] Differentially-expressed genes were 413 genes where 109
genes were for non-PD vs. Parkin, 233 genes for non-PD vs. PD and
335 genes for Parkin vs. PD. Particularly, 6 genes which had been
already known as PD-related gene were selected from 16 common genes
in the center of non-PD vs. Parkin vs. PD, and their genbank
accession numbers are listed in Table 4.
TABLE-US-00006 TABLE 4 Fold Change (log2 ratio) Genbank non-PD
non-PD Parkin Accession vs. vs. vs. No. Gene name (Gene symbol)
Parkin PD PD Function NM_152753 signal peptide, CUB domain,
EGF-like 3 2.0097 -1.6452 -3.6549 protein hetero- (SCUBE3)
homo-oligomerization NM_000584 interleukin 8 (IL8) -1.5026 -3.5819
-2.0792 angiogenesis/ cell motility NM_001677 ATPase,
Na.sup.+/K.sup.+ transporting, .beta.1 1.1420 -2.1375 -3.2796 ion
transport polypeptide (ATP1B1) NM_002546 tumor necrosis factor
receptor 1.2471 -1.6420 -2.8891 apoptosis/ superfamily, member 11b
(TNFRSF11B) inflammation response NM_004102 fatty acid binding
protein 3, muscle and -1.4108 1.7890 3.1998 phosphatidylcholine
heart (mammary-derived growth biosynthetic process inhibitor)
(FABP3) NM_001511 chemokine (C--X--C motif) ligand 1.quadrature.
-1.0412 -2.9751 -1.9339 chemotaxis/ (melanoma growth stimulating
activity, immune response .alpha.) (CXCL1)
[0103] In addition, 13 genes which have been known as Parkinson's
disease-related gene are selected from 56 genes in groups of non-PD
vs. Parkin and Parkin vs. PD, and their genbank accession numbers
are shown in Table 5. The genes do not include 16 genes which are
common in three groups.
TABLE-US-00007 TABLE 5 Fold Change Genbank (log2 ratio) Accession
non-PD Parkin No Gene name (Gene symbol) vs. Parkin vs. PD Function
NM_153262 synaptotagmin XIV (SYT14) 1.6903 -1.5708 membrane
trafficking NM_003667 leucine-rich repeat-containing G protein-
2.1567 -2.1239 G-protein signaling coupled receptor 5 (LGR5)
NM_003239 transforming growth factor, .beta.3 (TGFB3) 1.1171
-1.0345 cell growth/aging NM_002203 integrin .alpha.2 (ITGA2)
1.3831 -1.8448 cell adhesion NM_004101 coagulation factor II
(thrombin) receptor- 1.6712 -2.2182 G-protein signaling like 2
(F2RL2) NM_000794 dopamine receptor D1 (DRD1) 1.1496 -1.1496
G-protein signaling NM_006211 proenkephalin (PENK) 1.9852 -1.9852
neuropeptide signaling NM_004297 G protein .alpha.14 (GNA14) 2.5842
-2.5842 G-protein signaling NM_001122659 endothelin receptor type B
(EDNRB) 1.0755 -1.6347 G-protein signaling NM_021979 heat shock 70
kDa protein 2 (HSPA2) 1.0932 -1.4480 response to unfolded protein
NM_003043 solute carrier family 6, member 6 1.0595 -1.3053 amino
acid (SLC6A6) metabolic process NM_001628 aldo-keto reductase
family 1, member B1 1.1052 -1.3105 metabolic process (aldose
reductase) (AKR1B1) NM_005807 proteoglycan 4 (PRG4) 3.8210 -4.4772
cell proliferation
[0104] 2.3. Clustering Analysis and Result
[0105] After finally washing and staining, the image was scanned
with Affymetrix GeneChip.RTM. Human Gene 1.0 ST array using
Affymetrix Model 3000 G7 scanner. The image data was extracted by
Affymetrix Commnad Console software1.1. The raw excel file was used
for obtaining the expression extent data in the next step. The
expression data was obtained by Expression Console software version
1.1 (www.affymetrix.com). The data normalization was performed with
Robust Multi-Average (RMA) algorithm in Expression Console
software. The genes showing the increase of gene expression level
of at least two fold between the test group and the control group
were selected and used in the subsequent step.
[0106] The gene expression level of the selected genes was measured
with Hierarchical clustering in MEV (MultiExperiment Viewer)
software 4.0(http://www.tm4.org, TM4: a free, open-source system
for microarray data management and analysis. Biotechniques. 2003
February; 34(2):374-8.). To classify the genes as common gene
groups having similar expression pattern, K-mean Clustering
(http://www.tm4.org) was performed (A Soukas, P Cohen, N D Socci, J
M Friedman, Leptin-specific patterns of gene expression in white
adipose tissue, Genes Dev, 14, 963-80 (2000)). The K-mean
Clustering is a method used for classifying, based on their
patterns, the common gene expression groups having the genes of
similar expression pattern in hierarchical clustering analysis.
[0107] The genes which are differentially expressed were analyzed
biologically with Web-based DAVID (the Database for Annotation,
Visualization, and Integrated Discovery;
http://david.abcc.ncifcrf.gov/home.jsp, Systematic and integrative
analysis of large gene lists using DAVID Bioinformatics Resources.
(2009) Nat. Protoc. 4(1):44-57.). The genes were classified on the
basis of Gene ontology, Panther ontology database
(http://david.abcc.ncifcrf.gov/home.jsp).
[0108] Based on the similarity measured by the K-mean clustering
analysis between non-PD, PD and Parkin groups, the expressed genes
were classified. The expression pattern graphs were reorganized to
seven clusters (FIG. 5b).
[0109] FIG. 5a showed a result of Hierarchical Clustering by using
the signals of 413 genes which represent the difference in gene
expression between the groups of PA, PD, and Parkin. The result
confirmed the whole profile of clusters showing the difference in
gene expression between the groups of PA, PD, and Parkin, and the
expected several patterns of the clusters. FIG. 5b showed
classified clusters showing similar gene expression pattern, when 7
patterns of clusters were classified according to the result of
hierarchical clustering analysis. The number of pattern was
determined by the smallest optimized number which was obtained
after performing repetitive simulation with various pattern
numbers.
[0110] Specifically, the gene names and their genbank accession
number of Cluster 2, 3, 4, 5 and 6 were summarized in Tables
6a-6e.
TABLE-US-00008 TABLE 6a Cluster 2: Increase Genbank no Gene name
(non-PD < PD = Parkin) Gene symbol accession No 1 interleukin
.alpha.8 ITGA8 NM_003638 2 cathepsin H CTSH NM_004390 3 chemokine
(C-C motif) receptor-like 1 CCRL1 NM_178445
TABLE-US-00009 TABLE 6b Cluster 3: Increase Genbank no Gene name
(non-PD .ltoreq. PD < Parkin) Gene symbol accession No 1
transforming growth factor, .beta.3 TGFB3 NM_003239 2 dopamine
receptor D1 DRD1 NM_000794 3 G protein .alpha.14 GNA14 NM_004297 4
proenkephalin PENK NM_006211 5 proteoglycan 4 PRG4 NM_005807 6
leucine-rich repeat-containing LGR5 NM_003667 G-protein coupled
receptor 5 7 major histocompatibility HLA-DPA1 NM_033554 complex,
class II, DP .alpha.1
TABLE-US-00010 TABLE 6c Cluster 4: Decrease Genbank no Gene name
(non-PD .gtoreq. PD > Parkin) Gene symbol accession No 1 brain
expressed, X-linked 1 BEX1 NM_018476
TABLE-US-00011 TABLE 6d Cluster 5: Decrease Genbank no Gene name
(non-PD > PD = Parkin) Gene symbol accession No 1 interleukin 8
IL8 NM_000584 2 chemokine (C--X--C motif) ligand 6 CXCL6 NM_002993
(granulocyte chemotactic protein 2)
TABLE-US-00012 TABLE 6e Cluster 6: Increase Gene Genbank no Gene
name (non-PD .gtoreq. PD < Parkin) symbol accession No 1 signal
peptide, CUB domain, EGF-like 3 SCUBE3 NM_152753 2 heat shock 70
kDa protein 2 HSPA2 NM_021979 3 transforming growth factor, .beta.3
TGFB3 NM_003239 4 dopamine receptor D1 DRD1 NM_000794 5 G protein
.alpha.14 GNA14 NM_004297 6 proenkephalin PENK NM_006211 7
proteoglycan 4 PRG4 NM_005807 8 leucine-rich repeat-containing
G-protein coupled receptor 5 LGR5 NM_003667 9 reelin RELN NM_005045
10 endothelin receptor type B EDNRB NM_001122659 11 Integrin
.alpha.2 ITGA2 NM_002203 12 solute carrier family 6, member 6
SLC6A6 NM_003043 13 coagulation factor II (thrombin) receptor-like
2 F2RL2 NM_004101 14 cyclin-dependent kinase 6 CDK6 NM_001259 15
aldo-keto reductase family 1, member B1 (aldose reductase) AKR1B1
NM_001628 16 matrix metallopeptidease 8 MMP8 NM_002424 17 inhibitor
of DNA binding 1, dominant negative helix-loop- ID1 NM_181353 helix
protein 18 neurofilament, medium polypeptide NEFM NM_005382 19
ATPase, Na.sup.+/K.sup.+ transporting, .beta.1 polypeptide ATP1B1
NM_001677 20 tumor necrosis factor receptor superfamily, member 11b
TNFRSF11B NM_002546 21 tumor necrosis factor receptor superfamily,
member 10d, TNFRSF10D NM_003840 decoy with truncated death
domain
[0111] The increased pattern of gene expression was shown in
Clusters 2, 3, and 6, and the decreased pattern was shown in
Clusters 4 and 5. The gene data showing greatest linear-increase of
gene expression (Cluster 3) and the genes showing greatest
linear-decrease of gene expression (Cluster 4) could provide the
numerical values of severe Parkinson's disease and a guidance for a
search for the human biomarker diagnosing early-stage of
Parkinson's disease.
[0112] The gene expression result is described in detail
hereinafter.
[0113] Firstly, the genes which showed the linear decrease of gene
expression amount in the order of PA>PD>Parkin were Cluster 4
in Table 7 and FIG. 6.
TABLE-US-00013 TABLE 7 Gene name Function Genbank accession # brain
expressed, multicellular organismal NM_018476 X-linked 1
development // nervous system development // cell
differentiation
[0114] As shown in FIG. 6, gene expression amount of gene X-linked
1 (NM.sub.--018476) showed a linear decrease in PA, PD, and Parkin.
FIG. 6 is a pattern graph and Heat map showing a result of
Hierarchical Clustering of the genes separated with K-mean
Clustering Analysis.
[0115] The genes of Cluster 3 showed a linear increase of gene
expression amount in the order of PA<PD<Parkin, as shown in
Table 8 and FIG. 7.
TABLE-US-00014 TABLE 8 Gene name Function Genbank accession # major
histocompatibility complex, antigen processing and presentation of
NM_033554 class II, DP alpha 1 peptide or polysaccharide antigen
via MHC class II // immune response MHC class I polypeptide-related
antigen processing and presentation of NM_000247 sequence A peptide
antigen via MHC class I // response to stress // immune response //
cellular defense response // cell recognition // antigen processing
and presentation pancreatic lipase-related protein 3 lipid
catabolic process NM_001011709 secreted frizzled-related protein 4
signal transduction // embryo implantation NM_003014 // Wnt
receptor signaling pathway // cell differentiation Sestrin 3 cell
cycle arrest NM_144665 EGF-like repeats and discoidin I-like cell
adhesion // multicellular organismal NM_005711 domains 3
development //angiogenesis aldo-keto reductase family 1, member
prostaglandin metabolic process NM_003739 C3 (3-alpha
hydroxysteroid dehydrogenase, type II)
[0116] FIG. 7 is a result of clustering of the genes which showed a
linear increase of gene expression in non-PD, PD, and Parkin.
[0117] 2.4. Reprogramming of Functional Group of Human Biomarker
Candidate Among Non-PD vs. PD and Non-PD vs. Parkin
[0118] The functional groups among non-PD vs. PD and non-PD vs.
Parkin were reprogrammed, and the human biomarker candidates were
classified by using the genes obtained from Gene Ontology and
Panther database system (http://david.abcc.ncifcrf.gov/home.jsp)
(FIGS. 8a to 8d). FIGS. 8a to 8d represented the number of genes
which were reclassified as biologically-functional groups from the
genes having the gene expression level of at least two-fold between
the three groups of non-PD, PD, Parkin.
[0119] The biological categories included transcription factor,
nucleic acid binding, receptor, kinase, oxido-reduction protein,
signal molecule, cell adhesion molecule and the like.
[0120] The genetic functional groups which showed up-regulation
(FIG. 8a) or down-regulation (FIG. 8b) in Idiopathic PD patient
were compared with those of non-PD patient (control group). The
genetic functional groups which showed up-regulation (FIG. 8c) or
down-regulation (FIG. 8d) in Parkin (parkin deficiency) patient
were compared with those of non-PD patient (control group). These
graphs represented human biomarker candidate which were
re-classified according to the biological functions and notable
up-regulation or down-regulation in idiopathic PD patient and
parkin-deficiency PD patient.
[0121] 2.5. Genes Regulated Differentially in Non-PD, PD and Parkin
Patients Due to the Oxidative Stress
[0122] It has not been known which genes show selective sensitivity
to the oxidative stress and how those genes affect the cell.
PD-related genes which are differentially regulated by the
oxidative stress are analyzed in non-PD, PD and Parkin groups. The
genes regulated differentially by the oxidative stress were
classified again as a functional group from the genes showing the
gene expression level of at least two-fold between the groups of
non-PD, PD, and Parkin, and then the PD-related genes were selected
(refer to http://www.ncbi.nlm.nih.gov/pubmed/).
[0123] These groups included oxidoreductase, endoplasmic
reticulum/ubiquitin-like, exocytosis/membrane trafficking,
apoptosis/cell survival, structure/transport, translation,
nuclear/transcriptional, and cell cycle. The PD-related genes in
K-mean clustering 2 and 6 were classified again to change the
cluster number. The genes which were differentially expressed
between non-PD, PD and Parkin because of the oxidative stress are
summarized in Table 9.
TABLE-US-00015 TABLE 9 K-mean Putative Function clustering Group I:
oxidoreductase aldo-keto reductase family 1, member B1 (aldose
reductase) 6 (AKR1B1) Group II: endoplasmic
reticulum/ubiquitin-like dopamine receptor D1 (DRD1) 6 Group III:
exocytosis/membrane trafficking major histocompatibility complex,
class II, DP .alpha.1 3 (HLA-DPA1) synaptotagmin XIV (SYT14) 6
Group IV: apoptosis/cell survival actin, .alpha., cardiac muscle 1
(ACTC1) 2 clusterin (CLU) 2 transforming growth factor, .beta.3
(TGFB3) 6 Group V: structure/transport major histocompatibility
complex, class II, DP .alpha.1 3 (HLA-DPA1) transforming growth
factor, .beta.3 (TGFB3) 6 solute carrier family 6, member 6
(SLC6A6) 6 aldo-keto reductase family 1, member B1 6 (aldose
reductase) (AKR1B1) signal peptide, CUB domain, EGF-like 3 (SCUBE3)
6 Group VI: translation angiotensin II receptor, type 1 (AGTR1) 2
reelin (RELN) 6 G protein .alpha.14 (GNA14) 6 solute carrier family
6, member 6 (SLC6A6) 6 Group VII: nuclear/transcriptional integrin
.alpha.2 (ITGA2) 6 transforming growth factor, .beta.3 (TGFB3) 6
dopamine receptor D1 (DRD1) 6 Group VIII: cell cycle heat shock 70
kDa protein 2 (HSPA2) 6
[0124] Surprisingly, the selective gene expression of the groups in
Clusters 2, 3, and 6 increased linearly between non-PD, PD and
Parkin patients, and the groups belonged to AKR1B1
(oxidoreductase), DRD1 (endoplasmic reticulum/ubiquitin-like),
HLA-DPA1 and SYT14 (exocytosis/membrane trafficking), ACTC1, CLU
and TGFB3 (apoptosis/cell survival), HLA-DPA1, TGFB3, SLC6A6,
AKR1B1 and SCUBE (structure/transport), AGTR1, RELN, GNA14 and
SLC6A6 (translation), ITGA2, TGFB3 and DRD1
(nuclear/transcriptional), and HSPA2 (cell cycle). The genbank
accession numbers of the genes are summarized in the following
table.
TABLE-US-00016 Gene name Gene accession number AKR1B1 NM_001628
DRD1 NM_000794 HLA-DPA1 NM_033554 SYT14 NM_153262 ACTC1 NM_005159
CLU NM_001831 TGFB3 NM_003239 SLC6A6 NM_003043 SCUBE3 NM_152753
AGTR1 NM_000685 RELN NM_005045 GNA14 NM_004297 ITGA2 NM_002203
HSPA2 NM_021979
[0125] The obtained data can assist the understanding of
mitochondrial dysfunction and oxidative stress in Idiopathic and
Parkin-derived Parkinson's disease, and provide a useful guidance
for investigation of additional functional properties.
[0126] 2.6. Immortalization of Mesenchymal Stromal Cells Derived
from Adipose Tissue of Parkinson's Disease Patient with pGRN145
Including hTERT
[0127] The cells of non-PD, PD, Parkin in Example 1.1 were spread
again on the 24-well plate to reach 90 percent of confluence
without adding antibiotics on one day before transfecting. 50 .mu.L
of serum-free OPTI-MEM I Medium (Gibco BRL, Gaithersburg, Md.)
including 1 .mu.g of pGRN145 DNA (Geron Corporation, Menlo Park,
Calif., USA), and 50 .mu.L of OPT1-MEM I Medium including 2 mL of
LIPOFECTAMINE LTX Reagent (Gibco) were mixed and added to each
well, and replaced with new media after culturing at 37.degree. C.
for 24 hr. After 48 hours, the transfected cells were cultured in
media including Hygromycin-B (30 .mu.g/mL) for 2 to 3 weeks, and
the final concentration was reduced to be 10 .mu.g/mL. The clones
derived from one cell were selected.
[0128] The cell shapes of non-PD, PD, and Parkin cells belonging to
the selected clones were compared before immortalization, after
immortalization, 6-month culture and one-year culture with human
telomerase reverse transcriptase (hTERT). The result is described
in FIG. 9. In FIG. 9, non-PD (PA) represents the shape of cell
having Accession No. KCLRF-BP-00239(PA1) before and after
immortalization, PD is for the shape of cell having Accession No.
KCLRF-BP-00241(PD1) before and after immortalization, and Parkin is
for the shape of cell having Accession No. KCLRF-BP-00243(Pakin 1)
before and after immortalization.
[0129] The chromosomal structure of the cells obtained shortly
after the immortalization and after culturing the immortalized
non-PD, PD, and Parkin for a year were analyzed and shown in FIG.
10. The chromosomal structure of the cells before immortalization
was normal, and thus was not analyzed. From top to bottom in FIG.
10, non-PD (PA) represents the state of immortalized cell having
Accession Nos. KCLRF-BP-00239(PA1) and KCLRF-BP-00240(PA2), PD
represents the state of immortalized cell having Accession Nos.
KCLRF-BP-00241(PD1) and KCLRF-BP-00242(PD2), and Parkin represent
the state of immortalized cell having Accession Nos.
KCLRF-BP-00243(Pakin1) and KCLRF-BP-00244(Pakin2).
[0130] Specifically, the cell division at metaphase of mitosis was
restrained with colcemid (Gibco) Stoc solution. That is, the cells
were collected from the supernatant obtained by centrifuging at
1500 rpm, shocked with 0.075M KCl hypotonic, and fixed with the
addition of Canoy's fixative including methanol and acetic acid at
a mixing ratio of 3:1, and Giemsa staining GTG banding). The
prepared cell slide was analyzed with Karyotype Analysis program:
ChIPS-Karyo (Chromosome Image Processing System) (GenDix, Inc.
Seoul, Korea), and the analyzed result is shown in FIG. 10.
[0131] As shown in FIG. 10, the immortalized cell showed abnormal
nuclear type compared with the non-immortalized cell.
[0132] 2.7. Separation of Mitochondria from the Cultured Cell for
Mitochondria Complex I, II, IV and Citrate Synthase Assays
[0133] The non-PD, PD, and Parkin cells immortalized with hTERT
were washed with PBS and suspended in 10 mM Tris, pH 7.6 including
protease inhibitor cocktail. The cells were blocked with 1-mL
syringe, added with 1.5M sucrose and centrifuged at 600.times.g,
2.degree. C. for 10 minutes. Then, the supernatant were centrifuged
again at 14,000.times.g, 2.degree. C. for 10 minutes and the
obtained pellet were washed with protease inhibitor cocktail in 10
mM Tris (pH 7.6). The mitochondria pellets were re-suspended in 10
mM Tris (pH 7.6) including protease inhibitor cocktail and
subsequently preserved in ice before use.
[0134] Complex I assay: The activity of complex I was analyzed with
spectrometer at 600 nm by using 240 .mu.L reagent including 25 mM
potassium phosphate, 3.5 g/L BSA, 60 .mu.M DCIP, 70 .mu.M
decylubiquinone, 1.0 .mu.M antimycine-A, and 3.2 mM NADH, pH
7.8.
[0135] Namely, the obtained mitochondria sample (1 .mu.g/10 .mu.L)
was added to a buffer solution without NADH, incubated at
37.degree. C. for 3 minutes, and then added with 5 .mu.L of 160 mM
NADH. The absorbance was measured at 37.degree. C. for 5 minutes at
30 second-intervals, and after 5 minutes, and 2.5 .mu.L rotenone
(100 .mu.M of rotenone dissolved in 1 mM in dimethylsulfoxide and
10 mM Tris, pH 7.6) was added thereto. Then, the absorbance was
measured at 37.degree. C. for 5 minutes at 30 second-intervals. The
results are shown in FIG. 11.
[0136] Complex II assay: The activity of complex II was analyzed
with spectrometer at 600 nm with 240 .mu.L reagent including 80 mM
potassium phosphate, 1 g/L BSA, 2 mM EDTA, 0.2 mM ATP, 10 mM
succinate, 0.3 mM potassium cyanide, 60 .mu.M DCIP, 50 .mu.M
decylubiquinone, 1 .mu.M antimycine-A, and 3 .mu.M rotenone, pH
7.8.
[0137] Specifically, the obtained mitochondria sample (1 .mu.g/10
.mu.L) was added to a buffer solution without succinate and
potassium cyanide, incubated at 37.degree. C. for 10 minutes, and
then 204, of 1.5M succinate and 0.75 .mu.L of 0.1M KCN were added
thereto. The absorbance was measured at 37.degree. C. for 5 minutes
at 30 second-intervals, and BLANK was detected in the presence of 5
mM malonate. The result is shown in FIG. 11.
[0138] Complex IV assay: The activity of complex IV was analyzed
with spectrometer at 550 nm with 240 .mu.L reagent including 30 mM
potassium phosphate, 2.5 mM dodecylmaltoside, and 34 .mu.M
ferrocytochrome c, pH 7.4
[0139] Specifically, the obtained mitochondria sample (1 .mu.g/10
.mu.L) was added to a buffer solution and shortly after, the
absorbance was measured at 30.degree. C. for 5 minutes at 30
second-intervals, and BLANK was detected in the presence of 1 mM
KCN. The result is shown in FIG. 11.
[0140] Citrate synthase assay: The activity of citrate synthase was
analyzed with spectrometer at 412 nm with 240 .mu.L reagent (pH
7.5) including 50 mM Tris-HCl, 0.2 mM
5,5'-dithiobis-(2-nitrobenzoic acid), 0.1 mM acetyl-CoA and 0.5 mM
oxaloacetate.
[0141] Specifically, the obtained mitochondria sample (1 .mu.g/10
.mu.L) was added to a buffer solution without oxaloacetate, and
incubated at 30.degree. C. for 5 minutes. After adding by 2.5 .mu.L
of 50 mM oxaloacetate, the absorbance was measured at 37.degree. C.
for 5 minutes at 30 second-intervals. The result is shown in FIG.
11. This assay began with the addition of oxaloacetate, and for a
control group, water was added in the equal amount.
[0142] As represented in FIG. 11, the result of biochemical
analysis for mitochondrial respiration chain of immortalized cell
confirmed that the activities of PD and Parkin decreased compared
with Non-PD activity.
[0143] 2.8. Electron Microscopy Analysis
[0144] The non-PD, PD, and Parkin cells immortalized with hTERT
were washed with PBS and fixed with 0.1% glutaraldehyde and 4%
paraformaldehyde in PBS at 4.degree. C. for 2 hours. The cells were
collected and centrifuged at 2000.times.g, at 4.degree. C. and for
3 minutes to obtain pellets. The prepared pellets were re-suspended
in warm 1% agar and centrifuged at 2000.times.g at 4.degree. C. for
3 minutes to obtain the pellets again. Then, the pellets were
washed with PBS three times, and the cell pellets embedded with
agar were fixed again with 1% osmium tetroxide for 2 hours and
washed with PBS three times. The cell pellets embedded with agar
was dehydrated in ethanol and fixed again with Epon 812. The
ultrathin (70 nm) sections were collected on Formvar/carbon-coated
nickel grids, stained with 2.5% uranyl acetate for 7 minutes and
with lead citrate for 2.5 minutes, and then were observed with JEOL
JEM-1011 electron microscope. The results are shown in FIG. 12. The
comparison of mitochondrial shape of primary-cultured non-PD, PD
and Parkin cell and immortalized non-PD, PD and Parkin cell showed
that the mitochondria shape of non-PD (non-Parkinson's disease)
patient was normal, but those of PD (Idiopathic Parkinson's
disease) patient and Parkin (parkin-deficient Parkinson's disease)
patient were damaged gradually. This suggested the mitochondrial
damage is an important cause of Parkinson's disease.
[0145] 2.9. Western Blot Analysis
[0146] The cells (The primary-cultured mesenchymal stromal cells
derived from non-PD, PD, and Parkin patients were used in FIG. 13a,
and immortalized mesenchymal stromal cells derived from non-PD, PD,
and Parkin patients were used in FIGS. 13b, 13c, 14a, 15a, and 16a)
were washed with cold PBS and divided into lysis buffer (cell
signaling) and PMSF. The divided products were centrifuged at
15,000.times.g at 4.degree. C. for 20 minutes. The products were
analyzed quantitatively with Bradford reagent (Bio-Rad, Hercules,
Calif.). The protein which was used in the same amount as other
primary-cultured cells, and immortalized cells were loaded on
SDS-PAGE, transferred to PVDF membrane (Millipore), and blocked
with 5% non-fat dry milk in TBST. The proteins on the membrane were
detected by chemical luminescence with X-ray film using ECL-Plus
substrate (GE Healthcare, Buckinghamshire, USA). Antibodies of
Hsp25, Hsp60 and Hsp90 were obtained from Santa Cruz Biotechnology
(Santa Cruz, Calif., USA) and antibodies of DJ-1, P-mTOR, mTOR,
P-S6K, S6K, LC3-I, and LC3-II were obtained from Chemicon
(Temecula, Calif., USA). Prohibitin and .beta.-actin (Santa Cruz)
were used as internal control. The western blot analysis was
performed with National Institutes of Health image processing and
analyzing program (ImageJ, v1.38; http://rsb.info.nih.gov/ij/).
[0147] The obtained results are shown in FIGS. 13a-13c, 14a-14c,
15a-15b and 16a-16b. FIG. 13a represents the change in the gene
expression of mitochondrial markers, DJ-1, Hsp60, and Hsp25 in the
primary-cultured mesenchymal stromal cells derived from non-PD, PD,
and Parkin patients. FIG. 13b represents the change in the gene
expression of mitochondrial markers, Hsp60 in immortalized
mesenchymal stromal cells derived from non-PD, PD, and Parkin
patients. FIG. 13c represents the change in the gene expression of
mitochondrial markers, Hsp90 in immortalized mesenchymal stromal
cells derived from non-PD, PD, and Parkin patients. FIG. 14a
represents the change in the gene expression of autophagy marker,
mTOR, and S6K in immortalized mesenchymal stromal cells derived
from non-PD, PD, and Parkin patients. FIG. 14b and FIG. 14c are the
graphs showing the quantitative analysis of FIG. 14a. FIG. 15a
represents the change in the gene expression of autophagy marker,
mTOR, and S6K in immortalized mesenchymal stromal cells derived
from non-PD, PD, and Parkin patients. FIG. 15b is the graph showing
the quantitative analysis of FIG. 15a. FIG. 16a represents the
change in the gene expression of autophagy marker, LC3 (LC3-I,
LC3-II) in immortalized mesenchymal stromal cells derived from
non-PD, PD, and Parkin patients. FIG. 16b is the graph showing the
quantitative analysis of FIG. 16a.
* * * * *
References