KR101399696B1 - Method for screening agent inducing migration of stem cell into cartilage impairment - Google Patents

Abstract

The present invention relates to a method for screening a substance that promotes migration of stem cells into a cartilage damaged area. The screening method according to the present invention is a screening method in which heparin-conjugated fibrin gel (HCF) and heparin-conjugated fibrin gel (HCF) are bound to a cartilage injury site to allow the test substance to be slowly released, It is possible to select a substance that promotes the migration of stem cells to the cartilage damage site by measuring the movement of the cells in real time.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for screening a stem cell,

The present invention relates to a method and apparatus for binding a heparin-conjugated fibrin gel (HCF) to a cartilage damage site by binding heparin-conjugated fibrin gel (HCF) to the cartilage damage site, measuring the movement of the stem cell in real time, To a method for screening a substance capable of promoting the action of the substance.

There have been many attempts to solve the problem because joint cartilage damage is difficult to heal because the original tissue is not regenerated. These cartilage injuries are due to osteoarthritis, which causes traumatic defects or gradual destruction of articular cartilage tissue. However, it is difficult to regenerate the original cartilage as a supernatural bone and it is not well known before the molecular regulator of cartilage regeneration.

Conventional injured joint treatments include medication, autograft cartilage, bone marrow transplantation, and artificial joint replacement. Of these, conservative treatment such as drug treatment is limited to restoration of limited function to alleviate symptoms and to use for traumatic defect of articular cartilage Autologous osteochondral grafting causes donor damage due to osteo - cartilaginous sampling and has a disadvantage of limited harvesting. In addition, bone marrow transplantation for moderate osteoarthritis has the disadvantage of regenerating fibrous cartilage instead of the original cartilage tissue, the fibrous cartilage, and the clinical outcome is poor. In the current osteoarthritis, The life of the artificial joint also becomes a problem when it is performed.

In order to solve the above problems, recently, stem cells having an ability to self-replicate and differentiate into various tissues and having an advantage of easily collecting a large amount of cells without dysfunction of the donor are recognized as ideal cell sources for cell therapy, However, there is a lack of clear knowledge about factors and environment for cartilage formation.

Since adult mesenchymal stem cells can be obtained from the patient's own body, they do not interfere with the ethical problem of acquiring cells that have recently become a subject of interest, And it is recognized as a very useful cell source for the regeneration of musculoskeletal tissues. Most studies on adult stem cells that have been carried out to date have used mesenchymal stem cells cultured from bone marrow. In addition, adult stem cells are known to exist in most musculoskeletal tissues such as periosteum, adipose tissue and muscle.

Among these tissues, bone marrow is a typical source of stem cells. When bone cartilage is punctured, stem cells migrate directly to the defect site through bone marrow and cartilage damage sites. However, the important factor in determining the quality of regenerated cartilage is the number of stem cells that multiply and differentiate, so it is important to transfer many cells from the bone marrow to cartilage defect sites. In addition, since the transferred stem cells do not migrate to other parts but remain in the defective part and differentiate into cartilage at that part, a support containing cells and inducing differentiation is essential. It is desirable to expect cartilage regeneration by self-mobilization of stem cells, since it is impossible to localize cell treatment by cell-graft in a wide and general cartilage damage such as osteoarthritis.

If the stem cells can be moved to the cartilage damage site effectively, the cartilage can be effectively regenerated and the cartilage damage-related diseases can be treated. Therefore, a study for selecting a substance capable of promoting the migration of stem cells to the cartilage damage site need.

The present inventors investigated a method for screening a substance capable of promoting the movement of stem cells to a cartilage damage site. The heparin-conjugated fibrin gel (HCF) It was confirmed that the test substance was slowly released and the migration of stem cells could be observed in vivo by time, and the present invention was completed.

It is an object of the present invention to provide a method for screening a substance that promotes migration of stem cells into a cartilage damaged area.

The present invention relates to a method and apparatus for binding a heparin-conjugated fibrin gel (HCF) to a cartilage damage site by binding heparin-conjugated fibrin gel (HCF) to the cartilage damage site, measuring the movement of the stem cell in real time, The present invention provides a method of screening a substance that promotes the action of the substance.

The screening method according to the present invention is a screening method in which heparin-conjugated fibrin gel (HCF) and heparin-conjugated fibrin gel (HCF) are bound to a cartilage injury site to allow the test substance to be slowly released, The movement of the cells can be measured in vivo in real time to select a substance that promotes migration of the stem cells to the cartilage damage site.

FIG. 1 is a graph showing the effect of promoting the movement of mesenchymal stem cells after treatment with growth factors and chemokines (a: Boyden chamber assay, b: The number of transferred stem cells was quantified, and as a result, the number of transferred stem cells according to the concentration of c: PDGF-AA was determined.
FIG. 2 is a schematic diagram of an experiment for monitoring in vivo the process of transplanting the fluorescently labeled mesenchymal stem cells implanted intramammary with PDGF-AA into the cartilage damage site.
FIG. 3 is a graph showing the time-course of the movement of mesenchymal stem cells by PDGF-AA conjugated with heparin-conjugated fibrin gel (hereinafter referred to as HCF).
FIG. 4 is a graph showing the results of quantitative determination of changes in fluorescence of a cell-transplanted region (a) and a cartilage-damaged region (b) in the PDGF-AA releasing group.
FIG. 5 shows the results of stimulating the migration of human mesenchymal stem cells (HGF-PDGF-HD: PDGF-AA 50 ng, HGF-PDGF-LD 25 ng) (a) Comparison of migration of mesenchymal stem cells with 25ng of 50ng of PDGF-AA, b: Analysis of femur fluorescence image of immunosuppressed rats according to PDGF-AA concentration difference release, c: Histological analysis of cartilage site and detection of fluorescent marker cells)
FIG. 6 is a graph showing cartilage regeneration effect by simultaneous release of PDGF-AA and TGF-b at the cartilage damaged area (a: the degree of regeneration of cartilage after staining with paraffin tissue using saffranin- Results: b: ICRS visual score of each test group)
FIG. 7 is a graph showing the results of comparing the promoting effect of mesenchymal stem cell migration of test substances (CCL2, PDGF-BB) bound to HCF (a: bone fibrillary image of the femur taken 7 days after transplantation, b : Quantification of Fluorescence Detected at the Cartilage Damage Site).

The present invention relates to a method for preparing a test substance, comprising the steps of: 1) preparing heparin-conjugated fibrin gel (hereinafter referred to as HCF) with a test substance to prepare a binding substance of HCF and a test substance; 2) bonding the binding material of step 1) to a cartilage damage site; And 3) measuring, in real time, the movement of the stem cells to the cartilage damaged area by the adhered binding material of step 2); The present invention provides a method for screening a substance that promotes the migration of stem cells into a cartilage damaged area containing the cell.

Hereinafter, the screening method of the present invention will be described step by step.

In step 1), heparin-conjugated fibrin gel (hereinafter referred to as HCF) is bound to a test substance to prepare a binding substance of HCF and a test substance.

The fibrin is a kind of light protein and is an insoluble protein produced by the action of enzyme thrombin in fibrinogen in plasma. That is, fibrin forms a gel by enzymatic polymerization of fibrinogen in the presence of thrombin at room temperature. The fibrin gel thus formed is of great importance in the field of tissue engineering focusing on restoration of damaged tissues and organs. In particular, since the delivery of growth factor proteins is facilitated, fibrin gel can be used in the field of tissue regeneration such as bones, Many studies on gel have been carried out.

The heparin is a kind of glycosaminoglycan, and is used as an affinity ligand which binds to a certain class of heparin-binding proteins due to its highly negative charge property including a large amount of sulfate groups. Such properties are mainly used for chromatographic separation in protein separation, but recently, attention has been paid to the possibility of applying the heparin-binding protein to a specific site in a body by immobilizing it on a carrier of a protein preparation.

In the present invention, by binding HCF with the test substance expected to promote the migration of mesenchymal stem cells, the initial excessive release of the test substance can be prevented and sustained release compared with the use of conventional fibrin.

The test substance may be a chemokine or a growth factor, or a mixture thereof. The chemokine or growth factor may be selected from the group consisting of IL-8, Rantes, Monocyte inflammatory protein-3?, Stromal cell-derived factor-1?, BCA- ), CCL2 (CC motif ligand 2), CCL20 (chemokine CC motif ligand 2), CXCL16 (chemokine CXC motif ligand 16), CXCL12 (chemokine CXC motif ligand 12), endothelial growth factor (EGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor beta 1, insulin-like growth factor 1 (IGF- (Platelet Derived Growth Factor AB), PDGF-AA (Platelet Derived Growth Factor AA) and TNF-alpha (Tumor necrosis factor-alpha), preferably IL-8, CCL20, CXCL12, CCL2 , PDGF-AA, HGF, and IGF-1.

The step 2) is a step of bonding the binding material prepared in the step 1) to the cartilage damage site.

The binding substance refers to a substance in which HCF and a test substance are combined.

The adhesion refers to the formation of a hole leading to the bone marrow at the damaged cartilage area, and the mesenchymal stem cells are mixed with the matrigel in the same amount in the femoral bone marrow as the bone graft, and then the binding material is attached to the damaged cartilage area.

After the transplantation, the test substance may slowly be released to the cartilage damage site over a long period of time, and by releasing the test substance for promoting migration of the stem cell, Movement can be promoted.

The step 3) is a step of measuring in real time the movement of the stem cells to the cartilage damage site by the bonded substance.

The stem cells may be embryonic stem cells or adult stem cells. The adult stem cells are preferably selected from mesenchymal stem cells or stromal cells isolated from bone marrow, muscle, fat, cord blood, amniotic membrane or amniotic fluid, progenitor cells capable of differentiating into chondrocytes derived from the cells, Chondrocytes isolated from cartilage tissue, and cartilage cells separated from cartilage tissue. However, the present invention is not limited thereto. More preferably, the adult stem cells are selected from the group consisting of bone marrow-derived, adipose-derived, umbilical cord- Or more. These cells can be used singly or in a mixture of two or more kinds, and the cells can be isolated, proliferated, and differentiated into chondrocytes by any method known in the art.

The measurement of the migration of the stem cells can be performed by any method known in the art without limitation and can be performed by a person skilled in the art using a substance capable of generating a detectable signal such as a fluorescent substance, a ligand, a luminescent material, a microparticle, Can be measured. Preferably, the stem cells can be measured by fluorescence signal emission by labeling the moving stem cells with a fluorescent substance.

The fluorescent labeling substance may be, but not limited to, a fluorescent protein, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, fluorine synthioxyanate, etc. . Among them, fluorescence proteins known in the art can be used. For example, Cy5.5, GFP (Green Fluorescent Protein); Enhanced Green Fluorescent Protein (EGFP); RFP (Red Fluorescent Protein); mRFP (Monomeric Red Fluorescent Protein); DsRed (Discosoma sp. Red fluorescent protein); CFP (Cyan Fluorescent Protein); CGFP (Cyan Green Fluorescent Protein); YFP (Yellow Fluorescent Protein); AzG (Azami Green), HcR (HcRed, Heteractis crispa red fluorescent protein) and BFP (Blue Fluorescent Protein).

The adhered binding material may be slowly released over a period of 10 to 15 days, preferably about 14 days, and the migration of the stem cells to the cartilage damage site by the release of the binding substance may cause the fluorescence emitted from the stem cells And the fluorescence intensity is continuously decreased at the cell transplantation site and the fluorescence intensity is increased at the cartilage injury site, thereby evaluating the promotion effect of the stem cell to the cartilage damage site.

Hereinafter, the present invention will be described in detail with reference to Production Examples and Examples. However, the following Preparation Examples and Examples are illustrative of the present invention, and the content of the present invention is not limited by the following Production Examples and Examples.

Example  One. Liver  Selection of stem cell migration promoting factors

In order to identify candidates capable of promoting the migration of mesenchymal stem cells, mesenchymal stem cell migration promoting effects of IL-8, CCL20, CXCL12, CCL2, PDGF-AA, HGF and IGF-1 were confirmed. In order to confirm the effect of the candidate substance on the movement of mesenchymal stem cells, a Boyden chamber assay was performed and mesenchymal stem cells were stained to visualize cell migration.

The results are shown in Fig.

As shown in FIG. 1, when the growth factor PDGF-AA (Platelet Derived Growth Factor AA) was treated, the migration of mesenchymal stem cells was observed to the greatest extent, and the migration of mesenchymal stem cells by each candidate treatment was quantified The results showed that PDGF-AA was the most effective in promoting mesenchymal stem cell migration compared to other candidate substances. As shown in FIG. 1C, when 50ng / ml of PDGF-AA was treated, the mesenchymal stem cell migration was most promoted by the addition of PDGF-AA to the mesenchymal stem cells.

Example  2. Fibrin gel with covalently bound heparin ( Heparin -conjugated fibrin come ; Below, HCF )and Combined PDGF - AA Release and in vivo Liver  Stem cell migration monitoring

In vivo monitoring of transplantation of transplanted fluoroscopic mesenchymal stem cells into cartilage injury sites was performed. Immunosuppression A 1.5 × 1.5 × 1 mm lesion was made using a drill on the cartilage of the immunocompromised rats and a hole leading to the bone marrow was made and the fluorescently labeled mesenchymal stem cells inside the thigh bone marrow were transplanted in the same amount as Matrigel . In order to solve the existing problem of PDGF-AA being released at once, PDGF-AA was adhered to cartilage damaged area, and PDFG-AA and HGF were combined to allow the stem cell migration promoting substance to be slowly released over a long period of time. When HGF is used, the promoter of stem cell migration can be released slowly in the cartilage, unlike the case of using normal fibrin. Through this, it is possible to evaluate the promotion of stem cell movement effectively by observing the promotion of migration of stem cells in real time. The cultivation, transplantation and observation of stem cells are briefly shown in Fig.

The results of migration of mesenchymal stem cells by HCF-binding PDGF-AA are shown in FIG. 3 and FIG.

As shown in FIG. 3, it can be seen that the transplanted cells of HGF-only transplanted cells gradually disappear with the lapse of time. In the group transplanted with HGF and PDGF-AA, fluorescence- And gradually moved to the cartilage damaged area. In addition, as shown in FIG. 4, the fluorescence intensity of the cell-grafted region (a) and the cartilage damaged region (b) in the group in which PDGF-AA was released from HGF by grafting with HGF and PDGF- As a result of the quantification, the fluorescence intensity was continuously decreased for 14 days at the cell transplantation site (a), while the fluorescence intensity was rapidly increased for 14 days at the cartilage injury site (b). It was confirmed that PDGF-AA was not rapidly released by the binding of HGF and that the mesenchymal stem cells were allowed to migrate to the cartilage damage site with a gradual release over 14 days, The effect of promoting migration of stem cells was confirmed. Therefore, when HGF is conjugated to a candidate substance having a stem cell migration promoting effect, the candidate substance can be released over a long period of time in case of transplanting to a cartilage injury site, thereby enhancing the stem cell migration promoting effect of the candidate substance over time It is possible to effectively screen substances having the ideal release time and stem cell migration promoting effect among candidate substances promoting stem cell migration.

Example  3. PDGF - AA  Depending on concentration Liver  Assessment of promotion of migration of stem cells

In order to confirm whether the selective PDGF-AA binding to HGF can effectively promote mesenchymal stem cell migration, the promoting effect of mesenchymal stem cells was evaluated by varying the concentration of PDGF-AA. PDGF-AA was treated at 25 ng (low density group; LD) and 50 ng (high density group; HD) in cartilage damaged area and the migration characteristics of fluorescently labeled mesenchymal stem cells for 21 days were compared and analyzed. In addition, for further verification, immunostaining mice were killed at day 21 after stem cell transplantation, and the femur was extracted and fluorescence images were analyzed to observe the fluorescence. Sections were made with the paraffin block at the portion of the extracted cartilage where fluorescence was detected . To observe fluorescence, Cy5.5 was labeled and the labeled cells were confirmed by confocal microscopy.

The results are shown in Fig.

As shown in FIG. 5, cell migration was observed at 6 days and 9 days in the high-concentration treatment group than in the low-concentration treatment group of PDGF-AA combined with HGF. On the 21st day, however, more fluorescence was detected in the low concentration treatment group than in the high concentration treatment group (a). In addition, fluorescence image analysis of the extracted femur revealed more fluorescence in the cartilage damage site in the PDGF-AA low-concentration group (b), and this result was also the same in the histological analysis (c).

Example  4. PDGF - AA Wow TGF - Promoting the promotion of cartilage differentiation by simultaneous release of beta

In order to examine whether mesenchymal stem cells that have been promoted to cartilage site by PDGF-AA can actually promote cartilage differentiation at the cartilage damage site, the paraffin tissue was treated with saffranin-O at 21 days after mesenchymal stem cell transplantation Lt; / RTI > The degree of cartilage regeneration in the stained tissue was compared and expressed as the ICRS macroscopic score.

The results are shown in Fig.

As shown in FIG. 6, when a system in which PDGF-AA and TGF-b were simultaneously released was used, the cartilage regeneration effect was superior to that of the other groups. In the group 2, which was treated with only HCF-conjugated PDGF- (A), 21 days after the stem cell transplantation, the transferred stem cells were not differentiated into cartilage but scattered to other regions, so that no cells were observed. The ICRS visual scores showed that the best cartilage regeneration was achieved in Group 4, in which TGF-β was simultaneously released in HCF and PDGF-AA low-dose groups. Thus, it can be shown that PDGF-AA conjugated with HCF can promote mesenchymal stem cell migration to the cartilage damage site, and that the transferred mesenchymal stem cells can be differentiated into cartilage by TGF-β after 21 days from transplantation Respectively.

Example  5. Liver  Screening of stem cell migration promoting substances

In order to confirm the ability of HCF to promote mesenchymal stem cell migration in vivo in combination with the candidate group for promoting mesenchymal stem cell migration, the cells were transfected with CCL2, a chemokine, and PDGF-BB, a growth factor, in addition to PDGF- Promoting effect. For analysis, CCL2 or PDGF-BB was transplanted by the method of Example 2, and the femur was extracted on the 7th day after the operation to confirm the bone fluorescence image. In addition, fluorescence emitted from cartilage damage sites was quantitated.

The results are shown in Fig.

As shown in FIG. 7, it was confirmed that PDGF-BB was superior to CCL2 in promoting mesenchymal stem cell migration. As a result of fluorescence image quantification, PDGF-BB was found to promote stem cell migration more than twice as much as CCL2 Were analyzed numerically. As a result, it is possible to analyze the effect of promoting stem cell migration over time by inducing continuous release by suppressing the initial overexpression of the candidate group by combining HCF with a candidate substance for stem cell migration promoting substance, The fluorescence intensity was measured with time to evaluate the quantitative cell migration promoting effect, and thus it was confirmed that it can be usefully used for screening of stem cell migration promoting materials in vivo.

Claims (8)

1) preparing heparin-conjugated fibrin gel (HCF) and a test substance to prepare a binding material of HCF and a test substance;
2) adhering the binding material of step 1) to a cartilage damaged area of an animal other than a human; And
3) measuring in real time the movement of the stem cells to the cartilage damaged area by the adhesive material bonded in step 2)
The test substance may be selected from the group consisting of IL-8, Rantes, MIP-3β (monocyte inflammatory protein-3β), SDF-1α (Stromal cell-derived factor-1α), Endothelial growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor beta 1 (IGF-1), insulin-like growth factor 1 Screening for a substance that promotes migration of stem cells to cartilage damage sites, which is selected from the group consisting of Growth Factor AB, PDGF-AA, and TNF-alpha (Tumor Necrosis Factor-alpha) Way.
delete delete [3] The method according to claim 1, wherein the stem cells are embryonic stem cells or adult stem cells in step 3).
[Claim 5] The method according to claim 4, wherein the adult stem cells are at least one selected from the group consisting of bone marrow-derived, adipose-derived, umbrella-derived and umbilical cord blood-derived stem cells. Screening method.
The method according to claim 1, wherein the stem cell in step 3) is a stem cell labeled with a fluorescent substance.
The method of claim 6, wherein the fluorescent material is selected from the group consisting of fluorescent protein, fluorescein, isothiocyanate, rhodamine, picoeriterine, phycocyanin, allophycocyanin, and fluorine synthotioxyanate. Or more of the stem cells in the cartilage damaged area.
The method according to claim 1, wherein the test substance is released from the cartilage damage site for 10 to 15 days in the bonded binding material of step 3) .

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