U.S. patent application number 15/244284 was filed with the patent office on 2016-12-22 for mesenchymal stem cell attractant and method for attracting mesenchymal stem cell.
This patent application is currently assigned to Pias Corporation. The applicant listed for this patent is Osaka University, Pias Corporation. Invention is credited to Tadashi Furumoto, Kazuhiko Hamada, Yuta Inami, Yasufumi Kaneda, Keiko Kitamura, Akito Maeda, Koichi Nakaoji, Katsuto Tamai.
Application Number | 20160367616 15/244284 |
Document ID | / |
Family ID | 45066489 |
Filed Date | 2016-12-22 |
United States Patent
Application |
20160367616 |
Kind Code |
A1 |
Kitamura; Keiko ; et
al. |
December 22, 2016 |
MESENCHYMAL STEM CELL ATTRACTANT AND METHOD FOR ATTRACTING
MESENCHYMAL STEM CELL
Abstract
The present invention aims to provide a mesenchymal stem cell
attractant capable of attracting a mesenchymal stem cell; and
others. The present invention provides a mesenchymal stem cell
attractant containing at least one selected from the group
consisting of an extract of a Bodhi tree, an extract of the root
bark of Paeonia suffruticosa Andrews, an extract of Mallotus
philippinensis, an extract of Uncaria gambir, an extract of
Pashanbheda, and an extract of a plant belonging to the genus
Prunus.
Inventors: |
Kitamura; Keiko; (Kobe-shi,
JP) ; Inami; Yuta; (Kobe-shi, JP) ; Nakaoji;
Koichi; (Kobe-shi, JP) ; Hamada; Kazuhiko;
(Kobe-shi, JP) ; Maeda; Akito; (Suita-shi, JP)
; Furumoto; Tadashi; (Suita-shi, JP) ; Kaneda;
Yasufumi; (Suita-shi, JP) ; Tamai; Katsuto;
(Suita-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pias Corporation
Osaka University |
Osaka-shi
Suita-shi |
|
JP
JP |
|
|
Assignee: |
Pias Corporation
Osaka-shi
JP
Osaka University
Suita-shi
JP
|
Family ID: |
45066489 |
Appl. No.: |
15/244284 |
Filed: |
August 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13700844 |
Nov 29, 2012 |
9458429 |
|
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PCT/JP2011/055823 |
Mar 11, 2011 |
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15244284 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 36/47 20130101;
A61K 36/736 20130101; A61K 36/60 20130101; A61K 36/74 20130101;
A61K 36/185 20130101; A61P 43/00 20180101; A61P 17/02 20180101;
A61P 29/00 20180101; C12N 5/0663 20130101; A61K 36/65 20130101;
C12N 5/0662 20130101 |
International
Class: |
A61K 36/60 20060101
A61K036/60; A61K 36/65 20060101 A61K036/65; A61K 36/74 20060101
A61K036/74; A61K 36/736 20060101 A61K036/736; A61K 36/47 20060101
A61K036/47; C12N 5/0775 20060101 C12N005/0775; A61K 36/185 20060101
A61K036/185 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 1, 2010 |
JP |
2010-125905 |
Claims
1. A method for attracting a mesenchymal stem cell comprising
attracting a mesenchymal stem cell by applying an effective amount
of a mesenchymal stem cell attractant comprising at least one
selected from the group consisting of an extract of a Bodhi tree,
an extract of the root bark of Paeonia suffruticosa Andrews, and an
extract of Pashanbheda, to skin tissue, muscle tissue, cartilage
tissue or liver tissue.
2. The method for attracting a mesenchymal stem cell according to
claim 1, wherein the mesenchymal stem cell attractant is applied to
epidermal skin tissue.
3. The method for attracting a mesenchymal stem cell according to
claim 1, wherein the skin tissue has an injury site and the
mesenchymal stem cell attractant is applied to the injury site.
4. The method for attracting a mesenchymal stem cell according to
claim 1, wherein the mesenchymal stem cell attractant is applied to
the skin tissue, the muscle tissue, the cartilage tissue or the
liver tissue of humans.
5. The method for attracting a mesenchymal stem cell according to
claim 1, wherein the mesenchymal stem cell attractant is applied to
human skin.
6. The method for attracting a mesenchymal stem cell according to
claim 5, wherein the mesenchymal stem cell attractant is applied to
human epidermal skin tissue.
7. The method for attracting a mesenchymal stem cell according to
claim 1, wherein the mesenchymal stem cell attractant is applied to
the skin tissue, the muscle tissue, the cartilage tissue or the
liver tissue of animals other than humans.
8. The method for attracting a mesenchymal stem cell according to
claim 1 comprising attracting a mesenchymal stem cell in blood to
an injury site of skin tissue, muscle tissue, cartilage tissue or
liver tissue by applying an effective amount of a mesenchymal stem
cell attractant comprising at least one selected from the group
consisting of an extract of a Bodhi tree, an extract of the root
bark of Paeonia suffructicosa Andrews and an extract of Pashanbheda
to the injury site.
Description
TECHNICAL FIELD
[0001] The present invention relates to a mesenchymal stem cell
attractant and a method for attracting a mesenchymal stem cell.
BACKGROUND ART
[0002] As the stem cells that can be differentiated into various
tissue cells, embryonic stem cells (ES cells), which are the cells
derived from the undifferentiated zygote, somatic stem cells, which
are undifferentiated cells that are contained in differentiated
tissues, and the like are known.
[0003] Somatic stem cells are present in a variety of tissues in
the body, and examples thereof include mesenchymal stem cells
present in the bone marrow.
[0004] Mesenchymal stem cells are undifferentiated cells that can
be differentiated into cells belonging to mesenchymal tissues such
as bone, muscle, and fat, and are known to be capable of
differentiating also into ectodermal cells such as nerve cells and
endodermal cells such as liver cells.
[0005] Also, mesenchymal stem cells have drawn attention for their
ability to restore the function of a dysfunctional tissue by
differentiating into the cells in such a tissue. Specifically, for
example, bone marrow-derived mesenchymal stem cells have drawn
attention for their potential to differentiate into the cells in
inflamed tissues or damaged tissues under the influence of
differentiation inducers, which induce differentiation, by being
attracted to and accumulating in such tissues via the blood
stream.
[0006] Meanwhile, conventionally, various substances are known as
differentiation inducers that can differentiate mesenchymal stem
cells into a variety of tissue cells. For example, substances
containing PDGF-BB as the platelet-derived growth factor that can
differentiate mesenchymal stem cells into muscle tissue cells are
known (Patent Document 1).
CITATION LIST
Patent Document
[0007] Patent Document 1: International Publication No. WO
2005/063967
SUMMARY OF INVENTION
Technical Problem
[0008] However, the differentiation inducer of this kind has the
following problem: although the inducer is capable of
differentiating mesenchymal stem cells into specific tissue cells,
its ability to attract mesenchymal stem cells, for example, to
attract bone marrow-derived mesenchymal stem cells that are
circulating within the body via the blood stream to a specific
tissue in the body, is not necessarily satisfactory.
[0009] In view of the aforementioned problem and the like, the
present invention aims to provide a mesenchymal stern cell
attractant that can attract mesenchymal stem cells. Also, the
present invention aims to provide a method for attracting a
mesenchymal stem cell, which includes attracting a mesenchymal stem
cell by the aforementioned attractant.
SOLUTION TO PROBLEM
[0010] The mesenchymal stem cell attractant of the present
invention is characterized by containing at least one selected from
the group consisting of an extract of a Bodhi tree, an extract of
the root bark of Paeonia suffruticosa Andrews, extract of Mallotus
philippinesis, an extract of Uncaria gambir, an extract of
Pashanbheda, and an extract of a plant belonging to the genus
Prunus.
[0011] The method for attracting a mesenchymal stem cell according
to the present invention is characterized by attracting a
mesenchymal stem cell by the aforementioned attractant.
ADVANTAGEOUS EFFECTS OF INVENTION
[0012] The mesenchymal stem cell attractant of the present
invention has an effect of being capable of attracting a
mesenchymal stem cell.
BRIEF DESCRIPTION OF DRAWINGS
[0013] FIG. 1 is a diagram schematically illustrating the
chemotaxis assay.
[0014] FIG. 2 is a graph showing the results of the chemotaxis
assay using the attractant of Example 1.
[0015] FIG. 3 is a graph showing the results of the chemotaxis
assay using the attractant of Example 2.
[0016] FIG. 4 is a graph showing the results of the chemotaxis
assay using the attractant of Example 3.
[0017] FIG. 5 is a graph showing the results of the chemotaxis
assay using the attractant of Example 4.
[0018] FIG. 6 is a graph showing the results of the chemotaxis
assay using the attractant of Example 5.
[0019] FIG. 7 is a graph showing the results of the chemotaxis
assay using the attractant of Example 6.
[0020] FIG. 8 is a graph showing the results of the chemotaxis
assay using the negative control and the positive control.
DESCRIPTION OF EMBODIMENTS
[0021] Hereinafter, the embodiments of the mesenchymal stem cell
attractant of the present invention will be described.
[0022] The mesenchymal stem cell attractant according to the
present embodiments contains at least one selected from the group
consisting of an extract of a Bodhi tree, an extract of the root
bark of Paeonia suffruticosa Andrews, an extract of Mallotus
philippinensis, an extract of Uncaria gambir, an extract of
Pashanbheda and an extract of a plant belonging to the genus
Prunus.
[0023] The aforementioned extract of a Bodhi tree is obtained by
extracting a plant belonging to the family Tiliaceae with an
extraction solvent. Examples of the plant belonging to the family
Tiliaceae include Tilia platyphyllos Scop., Tilia cordata Mill.,
and Tilia europaea L, or other plants closely related to these
plants, Among them, as the plant belonging to the family Tiliaceae,
Tilia cordata Mill. is preferable because it has better ability to
attract mesenchymal stem cells. That is, as the aforementioned
extract of a Bodhi tree, an extract of Tilia cordata Mill, is
preferable.
[0024] The part of the aforementioned plant belonging to the family
Tiliaceae that is subjected to extraction is not particularly
limited, and examples of the part to be extracted include flowers,
fruits, and bark. Among them, as the part to be extracted, flowers
are preferable because they have better ability to attract
mesenchymal stem cells.
[0025] The aforementioned extract of the root bark of Paeonia
suffruticosa Andrews is obtained by extracting the root bark of
Paeonia suffruticosa Andrews with an extraction solvent.
[0026] The aforementioned extract of Mallotus philippinensis is
obtained by extracting Mallotus philippinensis belonging to the
family Euphorbiaceae with an extraction solvent.
[0027] The part of the aforementioned Mallotus philippinensis that
is subjected to extraction is not particularly limited, and
examples of the part to be extracted include leaves, branches,
trunks, bark, and roots. Among them, bark is preferable because it
has better ability to attract mesenchymal stem cells.
[0028] The aforementioned extract of Uncaria gambir is obtained by
extracting Uncaria gambir with an extraction solvent.
[0029] The part of the aforementioned Uncaria gambir that is
subjected to extraction is not particularly limited, and examples
of the part to be extracted include leaves and shoots. As the
aforementioned extract of Uncaria gambir, the extract obtained by
extracting both leaves and shoots with an extraction solvent is
preferable because it has better ability to attract mesenchymal
stem cells.
[0030] The aforementioned extract of Pashanbheda is obtained by
extracting one or two or more of Pashanbheda belonging to the genus
Bergenia stracheyi of the family Saxifragaceae with an extraction
solvent. Examples of Pashanbheda include Bergenia ligulata (Wall.)
Engl., Bergenia stracheyi (Hook. f. & Thoms) Engl., or Bergenia
ciliate (Haw.) Sternb. Among them, an extract obtained by
extracting Bergenia ligulata (Wall.) Engl. with en extraction
solvent is preferable because it has better ability to attract
mesenchymal stem cells.
[0031] The part of the aforementioned Pashanbheda that is subjected
to extraction is not particularly limited; however, rhizomes are
preferable because they have better ability to attract mesenchymal
stem cells.
[0032] The aforementioned extract of a plant belonging to the genus
Prunus is obtained by extracting a plant belonging to the genus
Prunus of the family Roaceae with an extraction solvent. Examples
of the plant belonging to the genus Prunus include Prunus speciosa,
Prunus jamasakura, Prunus sargentii, Prunus spachiana, Prunus
incisa, Prunus maximowiczii, Prunus.times.yedoensis, Prunus
nipponica, Prunus leveilleana, Prunus apetala, Prunus sub.
hirtella, Prunus lannesiana, and Prunus kanzakura, all belonging to
the subgenus Cerasus. Among them, as the plant belonging to the
genus Prunus, Prunus.times.yedoensis is preferable because it has
better ability to attract mesenchymal stem cells. That is, as the
aforementioned extract of a plant belonging to the genus Prunus, an
extract of Prunus.times.yedoensis is preferable.
[0033] The part of the plant belonging to the genus Prunus that is
subjected to extraction is not particularly limited, and examples
of the part to be extracted include flowers, roots, leaves, fruits,
and seeds. Among them, leaves are preferable because they have
better ability to attract mesenchymal stem cells.
[0034] One of the aforementioned extracts can be used alone, or a
mixture of two or more thereof can be used as the aforementioned
mesenchymal stem cell attractant.
[0035] Each of the aforementioned plant extracts can normally be in
the form of an extraction liquid obtained using the aforementioned
extraction solvent, a diluted solution of the extract, a
concentrate of the extract, or a dried product obtained by removing
the extraction solvent contained in the extract. Specifically, each
extract can be in the form of, for example, a solution, a paste, a
gel, and a powder.
[0036] Examples of the aforementioned extraction solvent include
water or an organic solvent such as an aliphatic monovalent alcohol
such as methanol, ethanol, and propanol; an aliphatic polyvalent
alcohol such as glycerin, propylene glycol, and 1,3-butylene
glycol; ketones such as acetone; diethyl ether, dioxane,
acetonitrile, and esters such as acetic acid ethyl ester; aromatic
compounds such as xylene, benzene, and toluene; and halogenated
alkyls such as chloroform.
[0037] One of the above extraction solvents can be used alone or a
mixture of two or more thereof can be used. The mixing ratio in the
mixture of the extraction solvents is not particularly limited and
is appropriately adjusted.
[0038] As the aforementioned extraction solvent, a hydrous
extraction solvent containing at least water is preferable. Also,
as the aforementioned extraction solvent an extraction solvent
containing an aliphatic alcohol such as an aliphatic monovalent
alcohol or an aliphatic polyvalent alcohol and water is more
preferable, and an extraction solvent containing an aliphatic
monovalent alcohol and water is even more preferable, and an
extraction solvent containing water and ethanol is most preferable
in view of better ability to attract mesenchymal stem cells.
[0039] As the aforementioned extraction solvent, specifically, for
example, an extraction solvent in which an aliphatic alcohol such
as an aliphatic monovalent alcohol or an aliphatic polyvalent
alcohol and water are mixed at a volume ratio of an aliphatic
alcohol water=7:3 to 3:7 is preferable.
[0040] More specifically, as the aforementioned extraction solvent,
an extraction solvent in which ethanol and water are mixed at a
volume ratio of ethane:water=7:3 to 3:7 is preferable.
[0041] The method of the aforementioned extraction is not
particularly limited, and a conventionally known, general
extraction method can be adopted. In extraction, the part of each
plant to be extracted can be used directly or after drying as the
extraction raw material. Also, normally, the amount of extraction
solvent is five to fifteen times (weight ratio) as much as the
amount of extraction raw material, and the extraction temperature
is 20.degree. C. to 80.degree. C. and the extraction time is two
hours to three days. After extraction, purification treatment such
as filtration, deodorization, and bleaching can be appropriately
performed as needed.
[0042] The concentration of each of the aforementioned extracts
contained in the aforementioned mesenchymal stem cell attractant is
not particularly limited. For example, the concentration is 0.1 to
5.0% by weight in terms of dry weight.
[0043] It is to be noted that the phrase "in terms of dry weight"
refers to converting the amount of extract into the weight of its
dried form, which is the residue obtained after removing the
extraction solvent from the extract.
[0044] Next, the embodiments of the method for attracting a
mesenchymal stem cell according to the present invention will be
described. The method for attracting a mesenchymal stem cell
according to the present embodiments is to attract a mesenchymal
stem cell by the aforementioned mesenchymal stem cell
attractant.
[0045] Mesenchymal stem cells are found in the mesenchymal tissue
cells, and they can differentiate not only into the cells of
mesodermal tissues such as cartilage, fat, and muscle, but also
into the cells of ectodermal tissues such as nerves and the cells
of endodermal tissues such as liver. Also, as the mesenchymal stem
cells to be attracted, bone marrow mesenchymal stem cells are
preferable because they can be comparatively easily collected from
the bone narrow and it has already been recognized that they can
also be present in blood.
[0046] According to the aforementioned method for attracting a
mesenchymal stem cell, mesenchymal stem cells can be attracted by
the aforementioned mesenchymal stem cell attractant in vitro, in
vivo, or in situ.
[0047] Specifically, for example, as the method for attracting a
mesenchymal stem cell in vitro, methods such as a method in which,
using an apparatus having a membrane having micropores that
penetrate in the direction of thickness, mesenchymal stem cells are
placed in one side of the membrane and the aforementioned
mesenchymal stem cell attractant is placed in the other side of the
membrane, whereby the mesenchymal stem cells are attracted to the
other side of the membrane over a certain period of time can be
performed.
[0048] Also, for example, as the method for attracting a
mesenchymal stem cell in vitro, methods such as a method in which
the mesenchymal cells inoculated onto a slide glass are partially
scraped off, and to the scraped area, a medium containing the
aforementioned mesenchymal stem cell attractant is added for
culturing, whereby the mesenchymal stem cells are attracted to the
scraped area can be performed. The degree of attraction of bone
marrow mesenchymal stem cells can be evaluated by confirming the
movement of mesenchymal stem cells in the scraped area.
[0049] Also, for example, as the method for attracting a
mesenchymal stem cell in vivo, methods such as a method in which an
aqueous gel containing the aforementioned mesenchymal stem cell
attractant is subcutaneously implanted in mice, and into these
mice, mouse bone marrow mesenchymal stem cells expressing Green
Fluorescent Protein (hereinafter, sometimes referred to as GFP) are
intravenously injected, and the mice are reared for a certain
period of time, whereby the bone marrow mesenchymal stem cells are
attracted to the gel can be performed. The degree of attraction of
bone marrow mesenchymal stem cells can be evaluated by measuring
the fluorescent intensity in the gel.
[0050] Also, for example, as the method for attracting a
mesenchymal stem cell in vivo, methods such as a method in which
mouse bone marrow mesenchymal stem cells expressing GFP are
transplanted into the one marrow of injury model mice, and at the
same time, the aforementioned mesenchymal stem cell attractant is
applied to the injury site in the mice, whereby the bone marrow
mesenchymal stem cells derived from the GFP mice are attracted to
the injury site can be performed. The degree of attraction of bone
marrow mesenchymal stem cells can be evaluated by measuring the
fluorescent intensity in the injury site.
[0051] Also, for example, as the method for attracting a
mesenchymal stem cell in situ, methods such as a method in which
the mesenchymal stem cell attractant is applied to a specific
tissue, whereby bone marrow mesenchymal stem cells are attracted to
the tissue can be performed. More specifically, for example,
methods such as a method in which the mesenchymal stem cell
attractant is applied to the skin epidermal tissue, whereby bone
marrow mesenchymal stem cells present in blood are attracted to the
epidermal tissue can be performed.
[0052] The aforementioned methods for attracting a mesenchymal stem
cell can be applied to animals other than humans in situ. Also,
these methods can be non-therapeutically applied to humans.
[0053] Also, examples of the specific tissue in the in situ method
of attraction include not only the aforementioned epidermal tissue,
but also a variety of tissues such as a muscle tissue, a cartilage
tissue, and a liver tissue.
[0054] In the aforementioned methods for attracting a mesenchymal
stem cell, the aforementioned mesenchymal stem cell attractant can
be used after dilution. Solutions used for dilution are not
particularly limited, and for example, water, physiological saline,
and a culture medium for mesenchymal cells can be used. No
particular limitation is imposed on the concentration of the
extract in the diluted solution obtained by diluting the
mesenchymal stem cell attractant when the attractant is used;
however, the concentration is preferably 0.00001 to 0.05% by weight
in terms of dry weight. It is advantageous that the concentration
of the extract is 0.00001%) by weight or in terms of dry weight be
that way excellent ability to further attract a mesenchymal stem
cell is achieved. Also, the concentration of the extract is
preferably 0.05% by weight or less because the toxicity to
mesenchymal stem cells can be further reduced.
[0055] Specifically, in the aforementioned methods for attracting
the mesenchymal stem cell, the attractant containing an extract of
a Bodhi tree is used preferably at a concentration of 0.00001 to
0.01% by weight, more preferably at a concentration of 0.0001 to
0.01% by weight in terms a dry weight.
[0056] Also, the attractant containing an extract of the root bark
of Paeonia suffruticosa Andrews is used preferably at a
concentration of 0.00001 to 0.01% by weight, more preferably at a
concentration of 0.0001 to 0.01% by weight in terms of dry
weight.
[0057] Also, the attractant containing an extract of Mallotus
philippinesis is used preferably at a concentration of 0.00001 to
0.01% by weight, more preferably at a concentration of 0.0001 to
0.01% by weight in terms of dry weight.
[0058] Also, the attractant containing an extract of Uncaria gambir
is used preferably at concentration of 0.0001 to 0.05% by weight,
more preferably at a concentration of 0.001 to 0.01% by weight in
terms of dry weight.
[0059] Also, the attractant containing an extract of Pashanbheda is
used preferably at a concentration of 0.00001 to 0.01% by weight,
more preferably at a concentration of 0.0001 to 0.01% by weight in
terms of dry weight.
[0060] Also, the attractant containing an extract of a plant
belonging to the genus Prunus such as an extract of
Prunus.times.yedoensis is used preferably at a concentration of
0.0001 to 0.05% by weight, more preferably at a concentration of
0.001 to 0.01% by weight in terms of dry weight.
[0061] The mesenchymal stem cell attractant and the method for
attracting a mesenchymal stem cell according to the present
embodiments are illustrated as above; however, the present
invention is not limited to the mesenchymal stem cell attractant
and the method for attracting a mesenchymal stem cell exemplified
as above. Also, in the present invention, various embodiments
adopted for general mesenchymal stem cell attractants and methods
for attracting a mesenchymal stem cell can be adopted so long as
the effect of the present invention is not impaired.
EXAMPLES
[0062] Next, the present invention will be described in further
detail with reference to Examples; however, the present invention
is not limited to these Examples.
[0063] First of all, as will be shown below, the mesenchymal stem
cell attractants composed of only respective extracts were produced
by preparing respective extracts. The detail of the production will
be described.
Example 1
[0064] As the extract of a Bodhi tree of Example 1, an extraction
liquid of Tilia cordata Mill. was prepared. In detail, to 100 g of
finely crushed dried flowers of Tilia cordata Mill., 1 L of a 50%
by volume aqueous solution of ethanol was added, and extraction
operation was performed at room temperature (20.degree.C.) for
three days, followed by filtration treatment. Further, the filtrate
was dried under reduced pressure to give a dried product, which was
diluted with 1,3-butylene glycol to prepare an extraction liquid of
Tilia cordata Mill. The resulting extraction liquid of a Bodhi tree
was found to contain 0.45% by weight of dried product according to
the calculation from the dry weight after removing the extraction
solvent by drying under reduced pressure.
Example 2
[0065] As the extract of the root bark of Paeonia suffruticosa
Andrews of Example 2, an extraction liquid of the root bark of
Paeonia suffruticosa Andrews was prepared. In detail, to 100 g of
finely crushed dried root bark of Paeonia suffruticosa Andrews, 1 L
of a 50% by volume aqueous solution of ethanol was added, and
extraction operation was performed at room temperature (20.degree.
C.) for three days, followed by filtration treatment. Further, the
filtrate was dried under reduced pressure to give a dried product,
which was diluted with 1,3-butylene glycol to prepare an extraction
liquid of the root bark of Paeonia suffruticosa Andrews. The
resulting extraction liquid of the root bark of Paeonia
suffruticosa Andrews was found to contain 0.90% by weight of dried
product according to the calculation from the dry weight after
removing the extraction solvent by drying under reduced
pressure.
Example 3
[0066] As the extract of Mallotus philippinensis of Example 3, an
extraction liquid of Mallotus philippinensis was prepared. In
detail, to 200 g of finely crushed dried bark of Mallotus
philippinensis Mueller-Argoviensis, 2 L of a 50% by volume aqueous
solution of ethanol was added, and extraction operation was
performed for two days while maintaining the temperature at 60 to
80.degree. C., followed by filtration treatment. Further, the
filtrate was dried under reduced pressure to give a dried product,
which was diluted with 1,3-butylene glycol to prepare an extraction
liquid of Mallotus philippinensis. The resulting extraction liquid
of Mallottus philippinensis was found to contain 0.20% by weight of
dried product according to the calculation from the dry weight
after removing the extraction solvent by drying under reduced
pressure.
Example 4
[0067] As the extract of Uncaria gambir of Example 4, an extraction
liquid of Uncaria gambir was prepared. In detail, to 100 g of
finely crushed dried leaves and shoots of Uncaria gambir Roxburgh,
2 L of a 50% by volume aqueous solution of ethanol was added, and
extraction operation was performed for three hours, while
maintaining the temperature at 50 to 70.degree.0 C. while stirring,
followed by filtration treatment. Further, the filtrate was dried
under reduced pressure to give a dried product, which was diluted
with 1,3-butylene glycol to prepare an extinction liquid of Uncaria
gambir. The resulting extraction liquid of Uncaria gambir was found
to contain 4.10% by weight of dried product according to the
calculation from the dry weight after removing the extraction
solvent by drying under reduced pressure.
Example 5
[0068] As the extract of Pashanbheda of Example 5, an extraction
liquid of Pashanbheda was prepared. In detail, to 200 g of finely
crushed dried rhizomes of Bergenia ligulata (Wall.) Engl., 3 Kg of
a 50% by volume aqueous solution of ethanol was added, and
extraction operation was performed at 50.degree. C. for eight hours
while stirring. The crude extract was cooled, filtered,
concentrated, and treated with a column filled with synthetic
adsorbents (trade name "Diaion HP-20", the product of Mitsubishi
Chemical Corporation). Subsequently, the column was washed with
water and eluted with a 30% by volume aqueous solution of ethanol.
The eluent thus obtained was dried under reduced pressure, and the
residue was dissolved again in 1,3-butylene glycol to prepare an
extraction liquid of Pashanbheda. The resulting extraction liquid
of Pashanbheda was found to contain 0.50% by weight of dried
product according to the calculation from the dry weight after
removing extraction solvent by drying under reduced pressure.
[0069] As the extract of the plant belonging to the genus Prunus of
Example 6, an extraction liquid of Prunus.times.yedoensis was
prepared. In detail, to 100 g of finely crushed dried leaves of
Prunus.times.yedoensis, 1 L of a 50% by volume aqueous solution of
ethanol was added, and extraction operation as performed at room
temperature (20.degree. C.) for three days while stirring, followed
by filtration treatment. Further, the filtrate was dried under
reduced pressure to give a dried product, which was diluted with
1,3-butylene glycol to prepare an extraction liquid of
Prunus.times.yedoensis. The resulting extraction liquid of
Prunus.times.yedoensis was found to contain 2.00% by weight of
dried product according to the calculation from the dry weight
after removing the extraction solvent by drying under reduced
pressure.
[0070] Subsequently each of the extracts was as the mesenchymal
stem cell attractant and evaluated and by a chemotaxis assay. FIG.
1 is a diagram schematically illustrating the evaluation method.
Hereinafter, the detail of the evaluation method described with
reference to FIG. 1.
[0071] <Chemotaxis Assay (Cell Attracting Test)>
[0072] The extracts produced in respective Examples were diluted to
0.01% by volume, 0.1% by volume, or 1% by volume to prepare test
samples. For dilution, Dulbecco's modified eagle's medium
[DMEM="FBS(-), P/S(-)"] was used. Here, FBS in the square brackets
indicates 10% fetal bovine serum and P/S indicates 100 units
penicillin and 0.1 mg/mL streptomycin. Also, the symbol (-)
indicates that the indicated substance is not added.
[0073] Meanwhile, as the negative control sample (hereinafter,
sometimes referred to as N.C.), DMEM "FBS(-), P/S(-)" was prepared,
and as the positive control sample (hereinafter, sometimes referred
to as P.C.), 20 ng/mL PDGF-BB (platelet-derived growth factor, the
product of Pepro Tech, Ltd.) was prepared.
[0074] Also, mouse bone marrow mesenchymal stem cells (hereinafter,
sometimes referred to as mMSC) were cultured to confluence and
harvested, and then suspended in 10% FBS/DMEM [P/S (-)] at
1.times.10.sup.7 cells/ml, whereby cell suspensions were
prepared.
[0075] Subsequently, Boyden chambers (the product of Neuro Probe,
Inc.) having a plurality of independent wells, in which the upper
well (P) and the lower well (Q) are separated by the membrane M as
shown in FIG. 1(a), were prepared. The chambers were set so that
any one of the test samples, the negative control sample, and the
positive control sample were tested in the same Boyden chamber, and
each sample was applied to each lower well of the chamber in an
amount of 28 .mu.l. It is to be noted that as the membrane of the
Boyden chamber, the trade name "Polycarbonate Membranes" (the
product of Neuro Probe, Inc., pore size 8 .mu.m) was adopted.
[0076] Subsequently, in the upper wells P of the Boyden chambers,
the aforementioned cell suspensions were inoculated in an amount of
50 .mu.l each, and then cultured for four hours under the
conditions of 37.degree. C. and 5% CO.sub.2 (see FIG. 1(b)).
[0077] After four hours of culture, as shown in FIG. 1(c), mMSC
that did not migrate was scraped off by the attached filter wiper
r, and only mMSC that had migrated to below the membrane was
stained with Diff-Quik stain (using the kit manufactured by Sysmex
Corporation).
[0078] Subsequently, the stained image was digitized and fed into a
computer, and in order to binarize the image into black and white,
the image was converted so that the part that was stained blue
appeared in white. Then, an average value of luminance within each
well was measured using the function of an image editing software
(the trade name "Photoshop"). By comparing the luminance of the
negative and positive control samples with that of each test
sample, the mMSC-attracting activity of the mesenchymal stem cell
attractant produced in each Example was evaluated.
[0079] The evaluation results of Examples 1 to 6 are each shown in
Example 2 to 7.
[0080] Also, as a referential experiment, a chemotaxis assay was
performed in a similar manner to the above by changing the
concentration of PDGF-BB used as the positive control sample. The
results thus obtained are shown in FIG. 8.
INDUSTRIAL APPLICABILITY
[0081] The mesenchymal stem cell attractant and method for
attracting a mesenchymal stem cell according to the present
invention can be suitably used for, for example, evaluating a
difference in the ability of each mesenchymal stem cell in various
tissues to accumulate (to be attracted) via migration.
[0082] Also, the mesenchymal stem cell attractant and method for
attracting a mesenchymal stem cell according to the present
invention can be suitably used in, for example, methods such as a
method in which the attractant is applied to a tissue in the body
by, for example, injecting or applying it to a specific tissue in
the body, whereby mesenchymal stem cells in blood that are
circulating in the body via the blood stream are attracted to and
accumulated in the specific tissue so that the mesenchymal stem
cells are, in the tissue, differentiated into the specific
tissue.
REFERENCE SIGNS LIST
[0083] P: Upper well, Q: Lower well, M: Membrane, and R: Filter
wiper
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