JP5820814B2 - Bone marrow stem cell attractant and bone marrow stem cell attracting method - Google Patents

Description

  The present invention relates to an attractant for bone marrow stem cells and a method for attracting bone marrow stem cells.

  Bone marrow stem cells are undifferentiated cells generated in the bone marrow and are known to be capable of differentiating into cells of various body tissues. In addition, it has been attracting attention as a tissue function that can be restored by differentiating into tissue cells in a tissue that has lost its function under the influence of a differentiation-inducing agent that induces differentiation.

  Specifically, bone marrow stem cells can be differentiated into tissue cells under the influence of a differentiation-inducing agent after moving from the bone marrow to the inflamed tissue or damaged tissue through the bloodstream, for example. It is attracting attention as.

  By the way, conventionally, various differentiation inducers capable of differentiating bone marrow stem cells into various cells are known. For example, fibroblast growth factor (Fibroblast Growth Factor) capable of differentiating bone marrow stem cells into cardiac muscle cells. Factor FGF) and platelet-derived growth factor (PGF) are known (Patent Document 1).

International Publication No. 2005/063967 Pamphlet

  However, although this type of differentiation inducer has the ability to differentiate bone marrow stem cells into predetermined tissue cells, it has the ability to attract bone marrow stem cells, specifically, for example, circulating in the body along the bloodstream. There is a problem that the performance of attracting bone marrow stem cells to a predetermined body tissue is not always sufficient.

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

  The attractant for bone marrow stem cells according to the present invention comprises at least one selected from the group consisting of deacetylcytochalasin C, a culture of Eupenicillium ludwigii, and a culture of Talaromyces trachyspermus. It is characterized by including.

The method for attracting bone marrow stem cells according to the present invention is characterized by attracting bone marrow stem cells non-therapeutically with the attractant.

  The attractant for bone marrow stem cells of the present invention has the effect of being able to attract bone marrow stem cells.

The figure showing 1-dimensional ¹ H-NMR spectrum data. The figure showing 1-dimensional ¹³ C-NMR spectrum data. The figure showing two-dimensional NMR (HH COSY) spectrum data. The figure showing two-dimensional NMR (HMQC) spectrum data. The figure showing two-dimensional NMR (HMBC) spectrum data. The figure showing MS spectrum data. 2 is a comparison table of one-dimensional NMR assignment data for the attractant of Example 2 and deacetylcytochalasin C. FIG. The figure showing the molecular structure of deacetylcytochalasin C. The figure which showed typically the mode of the cell attraction test in vitro. The figure which shows the result of the cell attraction test in vitro. The figure which shows the result of the cell attraction test in vitro. The graph which shows the result of the cell attraction test in vitro using the negative control sample and the positive control sample. The graph which shows the result of the cell attraction test in vitro using the fractionated fraction. The graph which shows the result of the cell attraction test in vitro using the fractionated fraction. The graph which shows the result of the cell attraction test in a biological body. The graph which shows the result of the cell attraction test in a biological body.

  Hereinafter, embodiments of the bone marrow stem cell attractant according to the present invention will be described.

  The attractant for bone marrow stem cells of this embodiment is at least one selected from the group consisting of deacetylcytochalasin C, a culture of Eupenicillium ludwigii, and a culture of Talaromyces trachyspermus. Is included.

The deacetylcytochalasin C is a compound having a molecular structure represented by the following formula (1).
The concentration of deacetylcytochalasin C contained in the bone marrow stem cell attractant is not particularly limited, and examples of the concentration include 0.001 to 100% by weight.

前記デアセチルサイトカラシンＣを含む骨髄幹細胞の誘引剤としては、製造が比較的容易であり骨髄幹細胞の誘引性能に優れるという点で、子のう菌類微生物に分類される黒彊病菌（メタリジウム アニソプリエ Metarhizium anisopliae）を培養することにより生成された生成物を含むものが好ましい。即ち、黒彊病菌（メタリジウム アニソプリエ Metarhizium anisopliae）の培養物が好ましい。

As an attractant for bone marrow stem cells containing the deacetylcytochalasin C, it is relatively easy to produce and has excellent attraction performance of bone marrow stem cells, so that it can be classified as a starter fungus microorganism (Metarhidium anisoprie Metarhizium Those containing products produced by culturing anisopliae) are preferred. That is, a culture of black rot fungus (Metarhizium anisopliae) is preferred.

  The culture of Eupenicillium ludwigii includes a product produced by culturing Eupenicillium ludwigii, which is classified as a starter fungus microorganism.

  The culture of Talaromyces trachyspermus includes a product produced by culturing Talaromyces trachyspermus classified as an irregular fungus microorganism.

  The above-mentioned culture of Eupenicillium ludwigii or Talaromyces trachyspermus is a crude culture containing cultured medium and cells, a diluted or concentrated product thereof, or a culture after using an extraction solvent. It can be used in the form of a culture extract obtained by subjecting the culture medium and bacterial cells to an extraction treatment. Preferably, it is preferably used in the form of the culture extract in that the concentration of the product produced by the culture is higher and the attracting performance of the attractant can be improved.

  Examples of the culture extract include an extract obtained with an extraction solvent, a diluted solution thereof, a concentrated solution thereof, or a dried product from which the extraction solvent has been removed. Specific examples include solution, paste, gel, and powder.

The concentration of the culture contained in the bone marrow stem cell attractant is not particularly limited, and is usually 0.001 to 20% by weight in terms of dry matter.
In addition, dry matter conversion is converting into the dry product of the culture product contained in the culture medium and microbial cell after culture | cultivation. The weight of the dried product is determined by the weight of the residue obtained by removing the culture medium, the bacterial cells, and the extraction solvent from the culture medium and the bacterial cells after culturing with a predetermined amount using the extraction solvent.

  Next, a method for producing the bone marrow stem cell attractant will be described.

The method for producing an attractant for bone marrow stem cells containing the deacetylcytochalasin C is not particularly limited, and the attractant may be, for example, deacetylcytochalasin C by culturing Metarhizium anisopliae. Can be produced.
Specifically, the bone marrow stem cell attractant containing deacetylcytochalasin C is, for example, cultured in the presence of a medium by cultivating black rot fungus (Metarhizium anisopliae) and subjecting the cultured medium and cells to an extraction treatment. In order to increase the concentration of deacetylcytochalasin C, if necessary, the culture extract can be further purified to produce a culture extract. More specifically, for example, it can be produced by the method described in the examples.

In the culture, a conventionally known general method can be employed. For example, a method of culturing using a solid medium, a method of culturing using a liquid medium, or the like can be employed.
The component which comprises the said culture medium is not specifically limited, As this component, the general organic compound, inorganic salt, etc. which can proliferate a microorganism can be used, for example.
In the culture, culture conditions such as culture time and culture temperature are appropriately adjusted.

As the extraction treatment method, for example, a solvent extraction method in which deacetylcytochalasin C is extracted from the culture medium and cells after culturing using an extraction solvent can be employed.
As the solvent extraction method, a conventionally known general method can be employed. Specifically, for example, a culture medium after culturing, bacterial cells, and an extraction solvent are mixed to prepare a suspension, and the suspension is separated by centrifugation, and a supernatant containing deacetylcytochalasin C is obtained. It is possible to adopt a method of collecting

As an extraction solvent in the solvent extraction method, water or an organic solvent can be used.
Specific examples of the organic solvent include aliphatic monohydric alcohols such as methanol, ethanol, butanol, and propanol; aliphatic polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; ketones such as acetone. Esters such as diethyl ether, dioxane, acetonitrile, and ethyl acetate; aromatics such as xylene, benzene, and toluene; alkyl halides such as chloroform, and the like.
The extraction solvent is used alone or in combination of two or more. The mixing ratio of the mixed extraction solvent to be mixed is not particularly limited and is appropriately adjusted.
As the extraction solvent, a mixed extraction solvent of acetone and water is preferably used in that deacetylcytochalasin C is more easily extracted.

A conventionally known general method can be employed as the purification method. Preferably, column chromatography such as high performance liquid chromatography (HPLC) is employed.
Specifically, as a method of the purification treatment, for example, the culture extract subjected to the extraction treatment as described above is fractionated into a plurality of fractions by high performance liquid chromatography (HPLC), and deacetylcytochalasin C A method of obtaining a fraction having a higher concentration can be employed.

In the method of fractionating into a plurality of fractions by the high performance liquid chromatography (HPLC), the concentration of deacetylcytochalasin C contained in the bone marrow stem cell attractant can be further increased. It is preferable to perform the same fractionation operation again for the fraction with increased sigma.
In addition, before performing the refinement | purification process by the said high performance liquid chromatography (HPLC), it is preferable to roughly refine | purify the culture extract which performed the extraction process as mentioned above. Specifically, for example, it is preferable to perform crude purification using a solid phase extraction cartridge in which a solid carrier is held by a resin syringe.

  As described above, a culture of Metarhizium anisopliae can be produced as an attractant for bone marrow stem cells containing deacetylcytochalasin C, and further purified deacetylcytochalasin C is produced. be able to.

  On the other hand, the attractant for bone marrow stem cells including a culture of Eupenicillium ludwigii or a culture of Talaromyces trachyspermus is Eupenicillium ludwigii or Talmyrus chy It can be produced by culturing by a conventionally known general method.

  In the production of an attractant for bone marrow stem cells containing a culture of Eupenicillium ludwigii or a culture of Talaromyces trachyspermus, the same method as the culture method described above can be employed.

In addition, in the production of an attractant for bone marrow stem cells containing the culture described above, an extraction process similar to the extraction process described above is performed in that the attractant's cell attracting performance can be improved. It is preferred to prepare a culture extract.
As the extraction solvent used for the extraction treatment, the aliphatic monohydric alcohol is preferable, and butanol is more preferable in that the attractant containing the culture can be more capable of attracting bone marrow stem cells.

  Subsequently, an embodiment of the method for attracting bone marrow stem cells of the present invention will be described. The method for attracting bone marrow stem cells according to the present embodiment is a method for attracting bone marrow stem cells using the bone marrow stem cell attractant.

Bone marrow stem cells are contained in the bone marrow tissue and can further differentiate within the bone marrow tissue or migrate from the bone marrow tissue to other body tissues along the bloodstream.
Examples of the bone marrow stem cells include bone marrow mesenchymal stem cells that can differentiate into cells of mesodermal tissue such as chondrocytes, adipocytes, and muscle cells, and hematopoietic stem cells that can differentiate into blood cells such as erythrocytes and leukocytes.
Among the bone marrow stem cells, bone marrow mesenchymal stem cells can differentiate not only into cells of mesodermal tissue but also into cells of endoderm tissue such as nerves and endoderm tissue such as liver. Bone marrow stem cells that are attracted in the attracting method of the present invention are preferably bone marrow mesenchymal stem cells in that they can be differentiated into more diverse cells after being attracted.

  In the method for attracting bone marrow stem cells, for example, bone marrow stem cells can be attracted in vitro or in vivo by the bone marrow stem cell attractant.

Specifically, in the method of attracting bone marrow stem cells in vitro, for example, using a device having a membrane (membrane) having micropores penetrating in the thickness direction, the bone marrow stem cells are arranged on one side of the membrane, For example, a method for attracting bone marrow stem cells to the other side of the membrane by placing the attractant for the bone marrow stem cells on the other side and waiting for a predetermined time can be performed. The degree of attraction of bone marrow stem cells can be evaluated by staining bone marrow stem cells attracted to the other side and measuring the degree of staining.
In addition, in the method of attracting bone marrow stem cells in vitro, for example, scraping a part of bone marrow stem cells seeded on a slide glass and adding a medium containing an attractant for bone marrow stem cells to the scraped portion and culturing. Thus, a method of attracting bone marrow stem cells to the scraped portion can be performed. The degree of attraction of bone marrow stem cells can be evaluated by confirming the proliferation of bone marrow stem cells in the scraped portion.
Since such an in vitro method of attracting bone marrow stem cells can be performed relatively easily, for example, a preliminary experiment was performed for the purpose of determining the optimum concentration of the attractant in the method of attracting bone marrow stem cells in vivo described below. can do.

On the other hand, in the method for attracting bone marrow stem cells in a living body, for example, an aqueous gel containing the above-mentioned bone marrow stem cell attractant is implanted under the skin of a normal mouse to express green fluorescent protein (hereinafter also referred to as GFP). A method for attracting bone marrow stem cells to an aqueous gel can be performed by injecting bone marrow stem cells of a mouse into a vein of the mouse and breeding the mouse for a predetermined period. The degree of attraction of bone marrow stem cells can be evaluated by measuring the intensity of fluorescence in the aqueous gel.
In the method of attracting bone marrow stem cells in vivo, for example, a bone marrow stem cell of a mouse expressing GFP is transplanted into the bone marrow of a wound model mouse, and the bone marrow stem cell attractant is applied to the wound part of the mouse. By doing so, a method of attracting bone marrow stem cells derived from GFP mice to the wound can be performed. The degree of attraction of bone marrow stem cells can be evaluated by measuring the intensity of fluorescence in the wound.

Furthermore, in the method for attracting bone marrow stem cells in a living body, for example, a method of applying a bone marrow stem cell attractant to a predetermined body tissue and attracting bone marrow stem cells to the tissue can be performed. More specifically, for example, a method of attracting bone marrow mesenchymal stem cells present in blood to the epidermal tissue by incorporating the bone marrow stem cell attractant into the epidermal tissue of the skin by application or the like. it can.
In addition, as a body tissue from which bone marrow stem cells are attracted in the attracting method in a living body, various tissues such as a muscle tissue, a cartilage tissue, and a liver tissue can be cited in addition to an epidermal tissue.

  The method for attracting bone marrow stem cells in a living body can be performed, for example, for the purpose of accumulating bone marrow stem cells in various tissues before differentiation into bone marrow stem cells.

  The method for attracting bone marrow stem cells can be performed in a living body of a human body or a non-human animal body. Preferably, it is performed non-therapeutically in a human body, specifically for cosmetic purposes, for example.

In the method for attracting bone marrow stem cells, the attractant for bone marrow stem cells can be diluted and used. The liquid for dilution is not particularly limited, and for example, water, physiological saline, bone marrow stem cell culture medium, and the like can be used.
The attractant of bone marrow stem cells containing deacetylcytochalasin C is used at a deacetylcytochalasin C concentration of 1-1000 μM by dilution, preferably at a deacetylcytochalasin C concentration of 1-100 μM. .
In addition, an attractant for bone marrow stem cells containing a culture of black rot fungus (Metarhizium anisopliae) is used, for example, at a concentration of 0.00001 to 0.2% by weight in terms of dry matter by dilution. Used at a concentration of 01-0.2% by weight.
Further, an attractant for bone marrow stem cells containing a culture of Eupenicillium ludwigii or a culture of Talaromyces trachyspermus is, for example, 0.00001 to 0.2% by weight in terms of dry matter by dilution. Is preferably used at a concentration of 0.01 to 0.1% by weight.

  The bone marrow stem cell attractant and bone marrow stem cell attractant of the present embodiment are as exemplified above, but the present invention is not limited to the above exemplified bone marrow stem cell attractant and bone marrow stem cell attractant. . Moreover, in this invention, the various form employ | adopted in the attractant of a general bone marrow stem cell and the method of attracting a bone marrow 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.

Example 1
As shown below, an attractant containing deacetylcytochalasin C was prepared.
That is, a black rot fungus (Metarhizium anisopliae) was cultured, and a crude culture containing the cultured medium and cells was obtained. A culture extract was prepared by subjecting this crude culture to an extraction treatment. Details will be described below.

First, in order to perform pre-culture of Metarhizium anisopliae, an inoculum extracted from a glycerol stock was cultured according to the following culture conditions.
-Pre-culture conditions: Temperature 28 ° C, 40 days-Pre-culture medium composition: LCA agar medium shown below
1g glucose
KH ₂ PO ₄ 1g
MgSO ₄ · 7H ₂ O 0.2g
KCl 0.2g
NaNO ₃ 2g
Bacto-yeast extract 0.2g
(Becton, Dickinson and Company)
Agar (Wako Pure Chemical Industries) 20g
Tap water 1L
NaOH 64.8mg
After pre-culture, the agar medium containing mycelium was punched with a straw. Meanwhile, 3.5 mL of sterilized tap water was dispensed into a 15 mL centrifuge tube. Five pieces of the punched agar medium were placed in a centrifuge tube, and the agar medium was crushed with a cotton swab to prepare a suspension.
Subsequently, as a medium for main culture, a main culture medium having the following composition is prepared in a 50 mL capacity centrifuge tube, and 1 mL of the above suspension is added to the main culture centrifuge tube. Main culture was performed.
・ Main culture conditions: Temperature 28 ° C., 2 weeks ・ Main culture medium composition: Solid medium shown below
Vegetable juice 10mL
(Product name “V8 Juice” manufactured by Campbell Soup Company)
Oatmeal 3g
(Snow Brand Milk Products (Quaker))
An 80 vol% acetone aqueous solution as an extraction solvent was added to the centrifuge tube after the main culture, and the crude culture was extracted with the extraction solvent.
Specifically, after adding the extraction solvent to the centrifuge tube, the centrifuge tube was vigorously shaken up and down to stir the contents, and allowed to stand at room temperature for 30 minutes. Thereafter, the centrifuge tube was centrifuged at 3500 rpm and 23 ° C. for 5 minutes, and about 15 mL of the supernatant after centrifugation was collected by decantation.
Subsequently, the supernatant was allowed to stand for 15 hours in a draft to evaporate the extraction solvent and concentrated, and then stored at −20 ° C. until the concentrated supernatant reached about 10 mL.
The stored supernatant was placed in a microtube, and a precipitate and a sample solution other than the precipitate were obtained by centrifugation at 13,000 rpm.
225.6 μL of the sample solution (corresponding to 200 μL of main culture broth) was dried to dryness with a centrifugal evaporator and stored at −20 ° C. The dried sample is dissolved in 100 μL of 80% by volume methanol aqueous solution and suspended for 30 minutes using a vortex mixer, and then the supernatant is obtained by centrifugation at 13,500 rpm, 4 ° C. for 5 minutes, followed by culture extraction A product was prepared and an attractant was produced.
The attractant contained 15% by weight of the dried product when calculated from the weight of the dried product after removing the solvent.
As described above, the attractant for bone marrow stem cells of Example 1 was produced. The fact that the attractant of Example 1 contains deacetylcytochalasin C is shown by the results of analyzing the attractant of Example 2 by nuclear magnetic resonance (NMR) and mass spectrometry (MS) methods.

(Example 2)
The attractant of Example 2 was produced by subjecting the attractant of bone marrow stem cells of Example 1 to further purification treatment. Specifically, the attracting agent for bone marrow stem cells of Example 1 was subjected to a crude purification treatment using a solid phase extraction cartridge, and after the treatment, elution with 90% by volume acetonitrile was performed at a higher speed. Purification was performed by liquid chromatography (HPLC). Details will be described below.
・ Rough purification (solid phase extraction cartridge)
Product name "Oasis HLB" (Waters) 200 mg / 6 cc
Subsequently, the crudely purified liquid was fractionated into 25 fractions by a purification process using HPLC (first fractionation operation). The HPLC conditions in the first fractionation operation are shown below.
-HPLC conditions (first fractionation operation)
Fractionation: Retention time after 3 minutes, fractionation every 10 minutes Column: C18 reverse phase column
(Product name: “Cosmosil 5C18-AR-II” 250 × 10 mm, manufactured by Nacalai Tesque)
Column temperature: 25 ° C
Mobile phase flow rate: 4 mL / min Mobile phase gradient conditions:
0 min-10% 1% formic acid aqueous solution / 50% water / 40% acetonitrile mixed solution 23 min-10% 1% formic acid aqueous solution / 90% acetonitrile mixed solution 28 min-acetonitrile (100%)
For each fraction obtained by the first fractionation operation, an in vitro cell attraction test (described later) is performed, and the fraction with the highest cell attraction performance (sixth) is further fractionated using HPLC. Performed (second fractionation operation). The HPLC conditions in the second fractionation operation are shown below.
-HPLC conditions (second fractionation operation)
Fractionation: Fractionation time 0 minutes or later, fractionation into a total of 25 fractions at 10 minute intervals
(Product name: “Cosmosil 5C18-AR-II” 250 × 4.6 mm, manufactured by Nacalai Tesque)
Column temperature: 25 ° C
Mobile phase flow rate: 0.85 mL / min Mobile phase gradient conditions:
0 min-10% 1% formic acid aqueous solution / 50% water / 40% acetonitrile mixed solution 20 min-10% 1% formic acid aqueous solution / 20% water / 70% acetonitrile mixed solution 22 min-acetonitrile (100%)
Among the fractions obtained by the above-mentioned second fractionation operation, the solvent (10th) having the best cell attracting performance in the in vitro cell attraction test described later is removed, and the solid (white powder) is removed. Thus, the attractant of Example 2 was produced.

About the white powder manufactured with the attractant of Example 2, a 1-dimensional and 2-dimensional nuclear magnetic resonance method (< ¹ > H-NMR, < ¹³ > C-NMR) and a deacetyl site by mass spectrometry (MS). Identified as calathin C. Details of the measurement method are shown below.

<Nuclear magnetic resonance method>
・ Measurement conditions Measuring device: JEOL Ltd. Device name “JNM-ECA”
Solvent: CD ₃ OD
One-dimensional NMR: ¹ H-NMR (600 MHz)
¹³ C-NMR (150 MHz)
Two-dimensional NMR: HH COSY (600MHz)
HMQC (600MHz)
HMBC (600MHz)
Each spectrum data of ¹ H-NMR, ¹³ C-NMR, H—H COSY, HMQC, and HMBC is shown in FIGS.

<Mass spectrometry>
・ Measuring conditions Measuring device: Applied Biosystems' device name “Voyager DE-STR”
Measurement method: Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry
(MALDI-TOF-MS (positive ion detection))
Matrix: α-cyano-4-hydroxycinnamic acid Spectral data is shown in FIG.

FIG. 7 shows a comparison table between the one-dimensional NMR assignment data of the substance obtained in Example 2 and the one-dimensional NMR assignment data of deacetylcytochalasin C. For the one-dimensional NMR assignment data of deacetylcytochalasin C in FIG. 7, Fujii, Y. et al., J. Nat. Prod., 2000, 63, 132-135 was referred to. In addition, those marked with * in FIG. 7 were judged to be indistinct signals due to the influence of the solvent.
Furthermore, what gave the position number to the molecular structure of deacetylcytochalasin C is shown in FIG.

(Example 3)
By culturing Eupenicillium ludwigii, a crude culture containing the cultured medium and cells was obtained. The crude culture was subjected to an extraction treatment to obtain a culture extract, and a bone marrow stem cell attractant was produced. Details thereof will be described below.
That is, first, the microbial cells taken out from the glycerol stock were planted in a gradient medium (malt extract agar medium (MA medium)) and pre-cultured for 1 week. Next, the cells after pre-culture are removed with a platinum loop and suspended in 5 mL of sterile physiological saline, and then the entire suspension is put into a 150 mL polypropylene flask containing a medium having the following composition. Inoculated and statically cultured (25 ° C., 12 days).
・ Medium composition Buckwheat flour 10g
SB solution 10mL (total 20g / flask)
-Composition of SB solution
Sodium tartrate 0.005%
KH ₂ PO ₄ 0.005%
MgSO ₄ · _7H 2 O 0.005%
FeSO ₄ · _7H 2 O 0.0005%
ZnSO ₄ · _7H 2 O 0.0005%
Yeast extract (Oriental Yeast Co., Ltd.) 0.01%
Water remainder Subsequently, the supernatant after separation by centrifugation was performed on 240 μL of the culture medium and bacterial cell mixture (crude culture) after culture using 100% butanol (100 μL) as an extraction solvent. Was lyophilized to remove the solvent.
Furthermore, 50 μL of methanol was added to the lyophilized product after extraction with butanol, and the mixture was suspended by stirring for 30 minutes with a vortex mixer, and then a supernatant was obtained by centrifugation at 13,500 rpm, 4 ° C. for 5 minutes. .
Thus, an attractant for bone marrow stem cells containing a cultured extract of Eupenicillium ludwigii was produced.
The attractant contained 2% by weight of the dried product as calculated from the weight of the dried product after removing the solvent.

Example 4
An attractant for bone marrow stem cells was produced in the same manner as in Example 3 except that Talaromyces trachyspermus was used in place of Eupenicillium ludwigii. The attractant contained 2% by weight of a dried product.

  Using the produced bone marrow stem cell attractant of each Example, evaluation was performed by an in vitro cell attraction test. FIG. 9 schematically shows the state of the evaluation method. Details of the evaluation method will be described below with reference to FIG.

<In vitro cell attraction test>
The concentration of deacetylcytochalasin C in Example 2 is 6.3 μM, 21 μM, and 63 μM, and the attractant in Examples 1, 3, and 4 is 0.01% by volume, 0.1% by volume. % Or 1% by volume to prepare a test sample.
Dulbecco's modified Eagle medium [DMEM “FBS (−), P / S (−)]] was used for dilution. Here, FBS in [] indicates 10% fetal bovine serum, and P / S indicates 100 units penicillin and 0.1 mg / mL streptomycin. Moreover, the symbol (-) indicates that no blending is performed.
On the other hand, DMEM “FBS (−), P / S (−)” is prepared as a negative control sample (hereinafter also referred to as NC), and as a positive control sample (hereinafter also referred to as PC). 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 mMSC) were cultured and collected until confluent, and suspended with 10% FBS / DMEM “P / S (−)” to 1 × 10 ⁷ cells / mL. It became cloudy and a cell suspension was prepared.
Next, a Boyden chamber (manufactured by Neuro Probe) having a plurality of independent wells, in which the well was partitioned into an upper well P and a lower well Q by a membrane M as shown in FIG. 9A, was prepared. . One kind of each test sample was set so that a negative control sample and a positive control sample could be tested in the same Boyden chamber, and 28 μL of each sample was applied to each lower well of the chamber. As the membrane of the Boyden chamber, a trade name “Polycarbonate Membranes” (pore size: 8 μm, manufactured by Neuro Probe) was used.
Subsequently, 50 μL of the cell suspension was seeded in the upper well P, and cultured for 4 hours under conditions of 37 ° C. and 5% CO ₂ (see FIG. 9B).
After culturing for 4 hours, as shown in FIG. 9 (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, and the image is converted to a white portion of the blue-stained portion so that the image is binarized into black and white. The measurement was performed using the function of editing software (trade name “Photoshop”). The mMSC attracting activity of the bone marrow stem cell attractant was assessed by comparing the brightness in the negative and positive control samples with the brightness in each test sample.

The evaluation results for the attractant of Example 1 are shown in FIG. Moreover, the evaluation result in the attractant of Example 2 is similarly shown in FIG.10 (b).
Similarly, the evaluation results of the attractants of Examples 3 and 4 are shown in FIGS. 11 (a) and 11 (b), respectively.
Further, as a reference experiment, a cell attraction test was performed in the same manner as described above by changing the concentration of the positive control sample PDGF-BB. The result is shown in FIG.

FIG. 13 shows a graph in which the vertical axis indicates the luminance of each fraction fractionated by the first fractionation operation in Example 2, the negative control sample, and the evaluation result of the test in the positive control sample.
Moreover, the graph which showed the brightness | luminance on the vertical axis | shaft about the evaluation result of the test in each fraction fractionated by 2nd fractionation operation in Example 2, a negative control sample, and a positive control sample is shown in FIG.

<Cell attraction test in mouse body>
Using the attractant (Metarhizium anisopliae culture extract) produced in Example 1 and the attractant (Deacetylcytochalasin C) produced in Example 2, a cell attraction test was performed in vivo. It was. The test was conducted by adopting the Matrigel plug method. Details of the test method are shown below.
The attractant of Example 1 has a concentration of 1.7% by weight (corresponding to a concentration of deacetylcytochalasin C of 0.03% by weight), and the attractant of Example 2 has a deacetylcytochalasin concentration of 80 μM. Each attractant and extracellular basement membrane gel (Matrigel) were mixed so as to obtain a C concentration, and placed in a resin tube.
Subsequently, the tube prepared as described above was transplanted to the back (left and right) of a 7-week-old C57BL / 6 mouse and reared for 2 weeks. After the breeding, the transplanted tube was taken out, the Matrigel in the tube was dissolved, and the cells in the tube were collected.
Furthermore, as a negative control sample, a similar tube containing only the above matrigel is prepared, and as a positive control sample, a tube containing fibroblast growth factor-2 (FGF-2) having a concentration of 500 ng / mL is prepared. did. And the cell attraction test was done similarly.
By measuring the fluorescence intensity in the cells attracted to the tube after the test, the cell attracting ability of the attractant of the example, the negative control, and the positive control was compared. Specifically, FITC-labeled lectin was bound to the cells collected from the tube, and the fluorescence intensity at a measurement wavelength of 485 nm and a control wavelength of 510 nm was measured. In each test, the number of mice was 4, and the average value of the obtained values (fluorescence intensity) was used as the evaluation result.

Meanwhile, bone marrow cells were killed by irradiating 7-week-old C57BL / 6 mice with 10 Gy (gray) X-rays. This mouse was transplanted with mouse bone marrow cells (4 × 10 ⁶ cells) labeled with green fluorescent protein (GFP).
Six weeks later, the percentage of GFP-positive cells contained in the bone marrow of transplanted mice was analyzed by flow cytometry (manufactured by Nippon Becton Dickinson, using the device name “BD FACSCantoII flow cytometer”). About 98% were GFP positive.
Further, in the same manner as described above, a cell attraction test was performed using tubes of a test sample, a negative control sample, and a positive control sample, and the collected cells were analyzed by flow cytometry. As a result, the cells contained in the matrigel of each sample were analyzed. More than 90% were GFP positive. From this, it was judged that 90% or more of the cells attracted in Matrigel were derived from bone marrow. Bone marrow cells are mainly composed of bone marrow stem cells.

  FIG. 15 and FIG. 16 show graphs showing the fluorescence intensity for the results of the cell attraction test. Specifically, FIG. 15 shows the results of the attractant (culture extract of Metarhizium anisopliae), negative control, and positive control of Example 1. FIG. 16 shows the results of the attractant (deacetylcytochalasin C), negative control, and positive control of Example 2.

  The bone marrow stem cell attractant and bone marrow stem cell attraction method of the present invention are used, for example, to attract bone marrow stem cells generated in the bone marrow to a predetermined body tissue before the bone marrow stem cells are differentiated in the predetermined body tissue. . That is, for example, it is preferably used for the purpose of attracting and accumulating bone marrow stem cells circulating in the body along the bloodstream by injecting or applying an attractant to a predetermined body tissue. .

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

Claims (2)

Deacetyl cytochalasin C, cultures Eupenishiriumu Rudowigii (Eupenicillium ludwigii), and attractants bone marrow stem cells, characterized in that it comprises at least one selected from the group consisting of a culture of Talaromyces Torakisuperumusu (Talaromyces trachyspermus) . Deacetyl cytochalasin C, cultures Eupenishiriumu Rudowigii (Eupenicillium ludwigii), and, by attractants bone marrow stem cells comprising at least one member selected from the group consisting of a culture of Talaromyces Torakisuperumusu (Talaromyces trachyspermus), non-therapeutic A method for attracting bone marrow stem cells, comprising attracting bone marrow stem cells.

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