JP2016028080A - Attractant for myeloid mesenchymal stem cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an attractant for myeloid mesenchymal stem cells which can attract myeloid mesenchymal stem cells.SOLUTION: The present invention provides an attractant for myeloid mesenchymal stem cells comprising at least one of Mallotus philippinensis extract, Tilia miqueliana extract, and Pashanbheda extract.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an attractant for mesenchymal stem cells.

Stem cells that have not yet differentiated and can differentiate into various tissue cells include embryonic stem cells (ES cells), which are cells in a state where fertilized eggs are not differentiated, and are not differentiated yet contained in differentiated tissues. Somatic stem cells, which are cells in a state, are known.
Somatic stem cells are present in various tissues in the body, and for example, mesenchymal stem cells present in bone marrow are known as somatic stem cells.

Mesenchymal stem cells can be differentiated into cells belonging to the mesenchymal system such as bone, muscle, and fat, but have not yet differentiated, and are also ectodermal cells such as nerve cells and endoderm such as liver cells. It is known that it can also differentiate into sex cells.
In addition, mesenchymal stem cells are attracting attention as being able to restore tissue function by differentiating into tissue cells in a tissue that has lost function. Specifically, for example, bone marrow-derived mesenchymal stem cells are attracted and accumulated in the bloodstream to inflamed or damaged tissues, and are affected by differentiation-inducing agents that induce differentiation. It has been attracting attention as being capable of differentiating into its tissue cells.

  By the way, conventionally, various differentiation inducing agents capable of differentiating mesenchymal stem cells into various tissue cells are known. For example, PDGF-BB as a platelet-derived growth factor (Platelet-Derived Growth Factor) is known. And the like which can differentiate mesenchymal stem cells into muscle tissue cells are known (Patent Document 1).

International Publication No. 2005/063967 Pamphlet

  However, although this type of differentiation inducer has the ability to differentiate mesenchymal stem cells into specific tissue cells, for example, bone marrow-derived mesenchymal stem cells circulating in the body in the bloodstream There is a problem that the ability to attract mesenchymal stem cells, such as attracting to a specific tissue in the body, is not always satisfactory.

  In view of the above-described problems and the like, an object of the present invention is to provide an attractant for mesenchymal stem cells that can attract mesenchymal stem cells. Another object of the present invention is to provide a method for attracting mesenchymal stem cells that attracts mesenchymal stem cells with the attractant.

  The attractant for mesenchymal stem cells according to the present invention is characterized by containing at least one of a bodaige extract, a button pipi extract, a coxnohagashi extract, an asenyaku extract, a pashambe extract, and a cherry extract. .

  The method for attracting mesenchymal stem cells according to the present invention is characterized by attracting mesenchymal stem cells by the attractant.

  The attractant for mesenchymal stem cells of the present invention has an effect that it can attract mesenchymal stem cells.

The figure which showed the mode in the cell attraction test typically. The graph which shows the result of a cell attraction test. The graph which shows the result of a cell attraction test.

Below, embodiment of the attractant of the mesenchymal stem cell of this invention is described.
In this embodiment, the mesenchymal stem cell attractant includes at least one selected from the group consisting of bodaiju extract, button pi extract, coxnohagashi extract, asenyaku extract, pashambe extract, and cherry extract. .

The body extract is obtained by extracting a plant belonging to the family Tiliaceae with an extraction solvent. Plants of the family Tiliaceae include tree swordfish ( Tilia platyphyllos Scop. ), Tree swordfish ( Tilia.cordata Mill. ), Linden tree ( Tilia.europaea L. ), or other related plants. Among these, the plant of the family Tiliaceae is preferably a coral mill ( Tilia.cordata Mill. ) In that it can attract more mesenchymal stem cells. That is, as the above-mentioned bodaige extract, a fuyubodaiji extract is preferable.

  The extraction site of the lindenaceae plant is not particularly limited, and examples thereof include flowers, fruits, and bark. Of these, flowers are preferred as the site to be extracted because they can attract more mesenchymal stem cells.

The button pi extract is obtained by extracting the root bark of Paeonia suffruticosa Andrews with an extraction solvent.

The above-mentioned Kusunohagashi extract is obtained by extracting with the extraction solvent Kusunohagashiwa ( Mallotus philippinensis ) belonging to Euphorbiaceae.

  The extraction site of the Kusonohagashi is not particularly limited, and examples thereof include leaves, branches, timber parts, bark, and roots. Among these, bark is preferable because it can attract more mesenchymal stem cells.

The asenyaku extract is obtained by extracting asenyaku ( Uncaria gambir ) with an extraction solvent.

  The extraction site of the asenyaku is not particularly limited, and examples thereof include leaves and young branches. As the Acacia catechu extract, an extract obtained by extracting both leaves and shoots with an extraction solvent is preferable in that it can attract more mesenchymal stem cells.

The Pashanbe extract is obtained by extracting one or more of Pashanbheda belonging to the genus Himalaya Yukinoshita with an extraction solvent. Examples of pashambe include Bergenia ligulata (Wall.) Engl. , Bergenia stracheyi (Hook. F. & Thoms.) Engl. Or Bergenia ciliata (Haw.) Sternb. Etc. Among these, those obtained by extracting Bergenia ligulata (Wall.) Engl. With an extraction solvent are preferable because they can attract more mesenchymal stem cells.

  The extraction site of the pashambe is not particularly limited, but a rhizome is preferable in that it can attract more mesenchymal stem cells.

The cherry extract is obtained by extracting rosaceous plants belonging to (Roaceae) Prunus (Prunus) extraction solvent. Examples of the plant belonging to the Prunus (Prunus), OOSHIMAZAKURA of cherry subgenus (Subgen.Cerasus) (Prunus.speciosa), cherry (Prunus.jamasakura), Prunus Sargentii (Prunus.sargentii), P. pendula f. Ascendens (Prunus.spachiana), Prunus incisa (Prunus.incisa), Prunus Maximowiczii (Prunus.maximowiczii), Yoshino cherry tree (Prunus x yedoensis), Prunus nipponica (Prunus.nipponica), Prunus Verecunda (Prunus.leveilleana), Choujizakura (Prunus.apetala), Prunus Subhirtella (Prunus.subhirtella), Satozakura ( Prunus.lannesiana ) and Kanzakura ( Prunus.kanzakura ). Among them, as a plant belonging to the genus Prunus , Yoshino cherry ( Prunus x yedoensis ) is preferable because it can attract more mesenchymal stem cells. That is, as the cherry extract, a Yoshino cherry extract is preferable.

The extraction site of a plant belonging to the genus Prunus is not particularly limited, and examples thereof include flowers, roots, leaves, fruits, and seeds. Of these, leaves are preferable because they can attract more mesenchymal stem cells.

  As the attractant for mesenchymal stem cells, one kind of the extract can be used alone, or two or more kinds can be used in combination.

  Each of the plant extracts described above can usually be in the form of an extract with the extraction solvent, a diluted solution thereof, a concentrated solution thereof, or a dried product from which the extraction solvent has been removed. Specifically, each extract can be in the form of, for example, a solution, a paste, a gel, or a powder.

Examples of the extraction solvent include water or aliphatic monohydric alcohols such as methanol, ethanol, and propanol; aliphatic polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; ketones such as acetone; And organic solvents such as esters such as dioxane, acetonitrile and ethyl acetate; aromatics such as xylene, benzene and toluene; and alkyl halides such as chloroform.
These extraction solvents may be used alone or in combination of two or more. The mixing ratio of the extraction solvent to be mixed is not particularly limited and is appropriately adjusted.

The extraction solvent is preferably a water-containing extraction solvent containing at least water.
In addition, the extraction solvent is more preferably an extraction solvent containing an aliphatic alcohol such as an aliphatic monohydric alcohol or an aliphatic polyhydric alcohol and water in that it can attract more mesenchymal stem cells. An extraction solvent containing a monohydric alcohol and water is more preferred, and an extraction solvent containing water and ethanol is most preferred.

Specifically, as the extraction solvent, for example, an aliphatic alcohol such as an aliphatic monohydric alcohol or an aliphatic polyhydric alcohol and water are mixed in a volume ratio of aliphatic alcohol: water = 7: 3 to 3: 7. The extraction solvent mixed in (1) is preferred.
More specifically, the extraction solvent is preferably an extraction solvent in which ethanol and water are mixed in a volume ratio of ethanol: water = 7: 3 to 3: 7.

  The extraction method is not particularly limited, and a conventionally known general extraction method can be employed. In the extraction, the extraction site of each plant can be used as it is or after drying as an extraction raw material. Further, the amount of extraction solvent is usually 5 to 15 times the weight of the extraction raw material (weight ratio), the extraction temperature is 20 ° C. to 80 ° C., and the extraction time is 2 hours to 3 days. After extraction, purification treatment such as filtration, deodorization, and decolorization can be appropriately performed as necessary.

It does not specifically limit as a density | concentration of said each extract contained in the attractant of the said mesenchymal stem cell, For example, 0.1-5.0 weight% in conversion of a dry matter is mentioned.
In addition, dry matter conversion is converting into the weight of the dry matter which is a residue remove | excluding the extraction solvent from the extract.

  In addition, as said each extract, what is marketed in the raw material for cosmetics, a food additive, etc. can be used, for example.

  Next, an embodiment of the method for attracting mesenchymal stem cells of the present invention will be described. The method for attracting mesenchymal stem cells according to the present embodiment is a method for attracting mesenchymal stem cells with the attractant for mesenchymal stem cells.

  Mesenchymal stem cells are contained in cells of mesenchymal tissues, and not only differentiate into cells of mesodermal tissues such as cartilage, fat, muscle, but also ectoderm tissues such as nerves, liver It can also differentiate into cells of endoderm tissues such as. Also, the mesenchymal stem cells to be attracted are preferably bone marrow mesenchymal stem cells in that they can be collected from the bone marrow relatively easily and can be present in blood.

In the method for attracting mesenchymal stem cells, the mesenchymal stem cells can be attracted by the mesenchymal stem cell attractant in vitro , in vivo , or in situ .

Specifically, for example, in an in vitro method for attracting mesenchymal stem cells, a device having a membrane (membrane) having micropores penetrating in the thickness direction is used, and mesenchymal stem cells are placed on one side of the membrane. The mesenchymal stem cell attractant may be disposed on the other side, and a method of attracting mesenchymal stem cells to the other side of the membrane after a predetermined time may be performed.
In addition, for example, in the method for attracting mesenchymal stem cells in vitro , a part of the mesenchymal cells seeded on the slide glass is scraped, and the mesenchymal stem cell attractant is contained in the scraped portion. A method of attracting mesenchymal stem cells to the scraped portion by adding a medium and culturing can be performed. The degree of attraction of bone marrow mesenchymal stem cells can be evaluated by confirming the proliferation of mesenchymal stem cells in the scraped portion.

In addition, for example, in an in vivo method for attracting mesenchymal stem cells, an aqueous gel containing the mesenchymal stem cell attractant is implanted subcutaneously in a normal mouse, and is also referred to as green fluorescent protein (hereinafter also referred to as GFP). The bone marrow mesenchymal stem cells of mice that have been expressed) are injected into the veins of the mice, and the mice are bred for a predetermined period of time, whereby a method of attracting the bone marrow mesenchymal stem cells to the gel can be performed. The degree of attraction of bone marrow mesenchymal stem cells can be evaluated by measuring the intensity of fluorescence in the gel.
In addition, for example, in an in vivo method for attracting mesenchymal stem cells, a bone marrow mesenchymal stem cell of a mouse expressing GFP is transplanted into the bone marrow of a wound model mouse, and the mesenchyme is transplanted into the wound portion of the mouse. By applying a systemic stem cell attractant, a method of attracting bone marrow mesenchymal stem cells derived from GFP mice to the wound can be performed. The degree of attraction of bone marrow mesenchymal stem cells can be evaluated by measuring the intensity of fluorescence in the wound.

In addition, for example, in the method for attracting mesenchymal stem cells in situ , a method for attracting bone marrow mesenchymal stem cells to the tissue by applying an attractant for mesenchymal stem cells to a specific tissue can be performed. . More specifically, for example, a method for attracting bone marrow mesenchymal stem cells present in blood to the epidermal tissue by applying the mesenchymal stem cell attractant to the epidermal tissue of the skin can be performed.

The method for attracting mesenchymal stem cells can be applied to animals other than humans in situ . It can also be applied non-therapeutically in humans.
The specific tissue in the in situ attracting method includes not only the above-described epidermal tissue but also various tissues such as muscle tissue, cartilage tissue, and liver tissue.

  In the method for attracting mesenchymal stem cells, the attractant for mesenchymal stem cells can be diluted and used. The liquid for dilution is not particularly limited, and for example, water, physiological saline, mesenchymal cell culture medium, or the like can be used. The concentration of the extract in the diluted solution obtained by diluting the attractant when using the mesenchymal stem cell attractant is not particularly limited, but is 0.00001 to 0.05 weight in terms of dry matter. % Is preferred. When the concentration of the extract is 0.00001% by weight or more in terms of dry matter, there is an advantage that the performance of attracting mesenchymal stem cells is more excellent. Moreover, it is preferable that the density | concentration of an extract is 0.05 weight% or less in conversion of a dry matter at the point that the toxicity to a mesenchymal stem cell may become a lower thing.

  The mesenchymal stem cell attractant and mesenchymal stem cell attracting method of the present embodiment are as described above, but the present invention is directed to the mesenchymal stem cell attractant and mesenchymal stem cell attractant described above. The method is not limited. Moreover, in this invention, the various form employ | adopted in the attractant of a general mesenchymal stem cell and the attracting method of a mesenchymal stem cell can be employ | adopted in the range which does not impair the effect of this invention.

  Next, although an Example is given and this invention is demonstrated in more detail, this invention is not limited to these.

  First, as shown below, an attractant for mesenchymal stem cells consisting only of each extract was produced by preparing each extract. Details thereof will be described.

Example 1
As the body extract of Example 1, a body juice extract was prepared. Specifically, 1L of a 50% ethanol aqueous solution is added to 100g of dried and finely crushed flowers of Tilia.cordata Mill , followed by extraction at room temperature (20 ° C) for 3 days, followed by filtration. went. Then, a dried product was obtained from the filtered solution by drying under reduced pressure, and this dried product was diluted with 1,3-butylene glycol to prepare a jujube extract. When calculated from the weight of the dried product after the extraction solvent was removed by drying under reduced pressure, this body extract contained 0.45% by weight of the dried product.

(Reference Example 1)
As a button pi extract of Reference Example 1, a button pi extract was prepared. In detail, 1L of 50 vol% ethanol aqueous solution was added to 100g of dried root bark of a button ( Paeonia suffruticosa Andrews ), followed by extraction at room temperature (20 ° C) for 3 days, followed by filtration. It was. Then, a dried product was obtained from the filtered solution by drying under reduced pressure, and this dried product was diluted with 1,3-butylene glycol to prepare a button pi extract. The button pi extract, when calculated from the weight of the dried product after the extraction solvent was removed by drying under reduced pressure, contained 0.90% by weight of the dried product.

(Example 2)
As the Kusunohagashi extract of Example 2, Kusunohagashi extract was prepared. Specifically, 200 L of 50% ethanol aqueous solution was added to 200 g of dried and finely crushed bark of Kusunohagashi ( Mallotus philippinensis Mueller-Argoviensis ), and extraction operation was carried out for 2 days while maintaining 60 to 80 ° C., followed by filtration. went. Then, a dried product was obtained from the filtered solution by drying under reduced pressure, and this dried product was diluted with 1,3-butylene glycol to prepare a Kusunohagashi extract. This Kusunohagashi extract, when calculated from the weight of the dried product after removing the extraction solvent by drying under reduced pressure, contained 0.20% by weight of the dried product.

(Reference Example 2)
An asenyaku extract was prepared as the asenyaku extract of Reference Example 2. Specifically, 2 L of 50 vol% ethanol aqueous solution was added to 100 g of dried and finely crushed leaves and young branches of Acacia yam ( Uncaria gambir Roxburgh ), and the extraction operation was performed for 3 hours while maintaining 50 to 70 ° C. with stirring. Filtration was performed. Then, a dried product was obtained from the filtered solution by drying under reduced pressure, and the dried product was diluted with 1,3-butylene glycol to prepare an asenyaku extract. This asenyaku extract was found to contain 4.10% by weight of the dried product as calculated from the weight of the dried product after the extraction solvent was removed by drying under reduced pressure.

(Example 3)
As the Pasambe extract of Example 3, a Pasambe extract was prepared. Specifically, 3 kg of a 50 vol% ethanol aqueous solution was added to 200 g of dried and finely crushed rhizomes of Pashambe ( Bergenia ligulata (Wall.) Engl. ), And extraction was performed at 50 ° C. for 8 hours while stirring. The crude extract was cooled and filtered, concentrated, and passed through a column packed with a synthetic adsorbent (trade name “Diaion HP-20” manufactured by Mitsubishi Chemical Corporation). And it washed with water, the eluate eluted with 30 volume% ethanol aqueous solution was dried under reduced pressure, and it redissolved in 1, 3- butylene glycol, and the Pashambe extract was prepared. When calculated from the weight of the dried product after the extraction solvent was removed by drying under reduced pressure, the pashambe extract contained 0.50% by weight of the dried product.

(Reference Example 3)
As a cherry extract of Reference Example 3, a Yoshino cherry extract was prepared. Specifically, 100 liters of dried Yoshino cherry ( Prunus x yedoensis ) leaves were added to 1 g of 50 vol% ethanol aqueous solution and stirred for 3 days at room temperature (20 ° C) with stirring. Went. Then, a dried product was obtained from the filtered solution by drying under reduced pressure, and this dried product was diluted with 1,3-butylene glycol to prepare a Yoshino cherry extract. This Yoshino cherry extract contained 2.00% by weight of the dried product when calculated from the weight of the dried product after the extraction solvent was removed by drying under reduced pressure.

  Next, each prepared extract was used as an attractant for mesenchymal stem cells and evaluated by a cell attraction test. FIG. 1 schematically shows the state of the evaluation method. The details of the evaluation method will be described below with reference to FIG.

<Cell attraction test>
Samples for testing were prepared by diluting the extract of each example to 0.01% by volume, 0.1% by volume, or 1% by volume. Dulbecco's modified Eagle medium [DMEM “FBS (−), P / S (−)]] was used for dilution. FBS represents 10% fetal bovine serum, and P / S represents 100 unit penicillin and 0.1 mg / mL streptomycin. Moreover, the symbol (-) indicates that no blending is performed.
In addition, DMEM “FBS (−), P / S (−)” is prepared as a negative control sample (hereinafter also referred to as NC), while a positive control sample (hereinafter also referred to as PC) is prepared. As a preparation, 20 ng / mL PDGF-BB (platelet-derived growth factor PEPRO TECH) was prepared.
In addition, mouse bone marrow mesenchymal stem cells (hereinafter also referred to as mMSCs) are cultured and collected until confluent, and suspended in 10% FBS / DMEM “P / S (−)” to 1 × 10 ⁷ cells / ml. It became cloudy and a cell suspension was prepared.
Next, each test sample was prepared using a Boyden chamber (manufactured by Neuro Probe) having a plurality of wells, in which an upper well P and a lower well Q are partitioned by a membrane M as shown in FIG. The negative control sample and the positive control sample were set to be tested in the same Boyden chamber, and 28 μl of each sample was applied to each lower well of the chamber. As a membrane of the Boyden chamber, a trade name “Polycarbonate Membranes” (pore size: 8 μm, manufactured by Neuro Probe) was adopted.
Subsequently, 50 μl of the cell suspension was seeded in the upper well P of the Boyden chamber partitioned by the membrane M, and cultured for 4 hours under conditions of 37 ° C. and 5% CO ₂ (FIG. 1B). See).
After culturing for 4 hours, as shown in FIG. 1 (c), the non-migrated mMSC was removed with the attached filter wiper R, and only the mMSC that had moved to the lower side of the membrane was subjected to Diff-Quick staining (using a Sysmex kit). ).
Then, the stained image is digitized and loaded into a computer, the image is binarized into black and white, the blue portion is converted to white, and the average value of the luminance in each well range is measured by the software “Photoshop”. By comparing with the luminance in the negative control sample and the positive control sample, the attracting activity of the mesenchymal stem cells of each Example was evaluated.

The evaluation results in each example are shown in FIGS.
In addition, as a reference experiment, a cell attraction test was performed in the same manner as described above by changing the concentration of PDGF-BB used in the positive control sample. The result is shown in FIG.

The mesenchymal stem cell attractant and mesenchymal stem cell attracting method of the present invention are for evaluating, for example, the difference in the ability to accumulate (or attract) through migration in each mesenchymal stem cell of various tissues. Can be suitably used.
The mesenchymal stem cell attractant and mesenchymal stem cell attracting method of the present invention can be applied to a tissue in the body by, for example, injecting the attractant into a specific tissue in the body. In order to differentiate mesenchymal stem cells into a specific tissue, it can be suitably used in a method for attracting and accumulating mesenchymal stem cells in blood circulating in the body by blood flow to the specific tissue.

  P: Upper well, Q: Lower well, M: Membrane, R: Filter wiper

Claims (2)

  An attractant for bone marrow mesenchymal stem cells for injection into epidermal tissue of the skin, comprising at least one of Kusunohagashi extract, Bodaiju extract, and Pashanbe extract.   An attractant for bone marrow mesenchymal stem cells for attracting bone marrow mesenchymal stem cells in blood to a wound site, comprising at least one of Kusonohagashi extract, Bodaiju extract, and Pashambe extract.