WO2014042292A1 - Composition comprising protein kinase c activator for promoting stem cell adhesion, and method for promoting stem cell adhesion - Google Patents

Abstract

The present invention relates to a composition comprising a protein kinase C activator for promoting stem cell adhesion and to a method for promoting a stem cell adhesion. The protein kinase C activator according to the present invention promotes the phosphorylation of focal adhesion kinase (FAK) which is a signal transduction molecule associated with cell adhesion capacity in stem cells, and increases the expression of paxillin, talin, vinculin and Rac1 which is a GTPase, and thus enables stem cells to effectively adhere to a damaged area of the myocardium.

Description

Stem cell adhesion promoting composition comprising protein kinase C activator and method for promoting adhesion of stem cells

The present invention relates to a composition for promoting stem cell adhesion comprising a protein kinase C activator and a method for promoting the attachment of stem cells.

The present invention also relates to a stem cell promoted adhesion by protein kinase C activator, and a pharmaceutical composition for treating ischemic heart disease comprising the same as an active ingredient.

In the 21st century, biotechnology proposes new solutions to food, environment, and health problems as the final goal of human welfare. Recently, stem cell therapy is emerging as a new chapter in the treatment of intractable disease. Previously, organ transplantation and gene therapy have been suggested for the treatment of intractable disease in humans. However, due to immunorejection reaction, lack of supply organs, vector development or lack of knowledge about disease genes, efficient practical use has been insufficient.

As an alternative to solve these problems, interest in stem cells has increased, and pluripotent stem cells with the ability to form all the organs through proliferation and differentiation are treated as well as most diseases, Parkinson's disease and various cancers. It has been suggested that it can be applied to a variety of treatments such as diabetes and spinal cord injury.

"Stem cell" refers to a cell that has the ability to self-replicate and differentiate into two or more new cells. Embryonic stem cells, induced pluripotent cells, and adults Stem cells (adult stem cells) and the like are the most actively studied. In the case of embryonic stem cells or induced pluripotent stem cells, there are still limitations in applying them to the clinic due to stability issues such as ethical issues and tumor formation. In the case of adult stem cells, clinical studies have been conducted in patients due to its stability, but in the case of neural stem cells and cord blood cells, adult cells are transplanted to themselves. Autograft is not yet possible and there is a limit to transplant the neural stem cells of others. Bone marrow or fat-derived mesenchymal stem cells are stem cells that are most actively studied in the clinic because they can be easily obtained from the human body.

Heart disease is one of the leading causes of death worldwide and one of the leading causes of death in recent years. Myocardial injury by myocardial infarction leads to irreversible loss of tissue and cardiac dysfunction, and the remaining cardiomyocytes in the damaged tissue are gradually replaced by fibroblasts trying to form scar tissue. Although medical and device-based therapies can ameliorate the symptoms of these heart diseases, the limitations of treatment appear in that they cannot reverse the loss of cardiomyocytes, the root cause of the problem. Therefore, there have been attempts to transplant embryonic stem cells or cardiomyocytes as a treatment for regenerating damaged cardiomyocytes.However, transplantation of cardiomyocytes has difficulty in cell acquisition, and in the case of embryonic stem cells, problems such as tumor formation and immunorejection are difficult. It is not yet known how to regenerate cardiomyocytes effectively.

Bone marrow-derived mesenchymal stem cells can differentiate into cardiomyocytes, endothelial cells and smooth muscle cells when externally stimulated, and several reports indicate that these mesenchymal stem cells can differentiate into myocardium in vivo and in vitro. Due to the fact that it contributes to myocardial regeneration, the transplantation of mesenchymal stem cells is expected to be a breakthrough treatment to heal myocardial injury and prevent heart failure after injury. However, the low survival rate of mesenchymal stem cells transplanted into injured myocardial tissue is a limitation of cell therapy. In fact, the survival rate of stem cells transplanted into the injured myocardium is known to be less than 1%, suggesting that the effectiveness of stem cell therapy, which has been clinically tried in myocardial infarction disease, is not greater than expected. It is believed that one of the causes of stem cell death after transplantation is due to the low adhesion between the transplantation site and the transplanted cells, which is believed to weaken the signaling mechanisms associated with cell survival and differentiation.

As described above, there are limitations due to the low survival rate of stem cells in treating stem cells for treating myocardial injury patients. However, there is no report on how to increase the adhesion of stem cells to overcome these limitations. Therefore, there is an urgent need for a method for increasing the survival rate of stem cells by increasing the adhesion of stem cells to overcome the existing limitation of stem cell therapy.

The inventors of the present invention, while applying the appropriate stimulation to the stem cells, while studying a method for increasing the adhesion of the stem cells, when the protein kinase C activator is treated to the stem cells, the adhesion and spreading capacity of the stem cells increases It was confirmed that the adhesion of stem cells in the implantation site, such as to complete the present invention.

It is an object of the present invention to provide a composition for promoting stem cell adhesion and a method for promoting stem cell adhesion using the same, comprising a protein kinase C activator as an active ingredient.

In addition, another object of the present invention is to provide a stem cell enhanced adhesion, treated with a protein kinase C activator.

In addition, another object of the present invention is to provide a pharmaceutical composition for treating heart disease, comprising as an active ingredient stem cells having increased adhesion ability treated with a protein kinase C activator.

In order to achieve the above object, the present invention provides a composition for promoting stem cell adhesion and stem cell adhesion promoting method comprising a protein kinase C activator as an active ingredient.

In another aspect, the present invention provides a stem cell enhanced adhesion ability, treated with a protein kinase C activator.

In another aspect, the present invention provides a pharmaceutical composition for treating ischemic heart disease comprising stem cells having increased adhesion ability treated with a protein kinase C activator as an active ingredient.

Protein kinase C activator according to the present invention promotes the phosphorylation of FAK (focal adhesion kinase), a signaling molecule associated with cell adhesion in stem cells, paxillin, Tallinn, vinculin and GTase By increasing the expression of Rac1 phosphorus, there is an excellent effect to effectively attach stem cells to the site of transplanted myocardial damage.

1 is a result of confirming the adhesion ability of mesenchymal stem cells according to PMA (phorbol myristate acetate) treatment according to PMA treatment concentration (20, 50, 100, 200, 500 nM) (a) and mesenchymal at 100nM PMA treatment Figure (b) shows the adhesion of stem cells (* p <0.05 vs. control).

Figure 2 is a diagram showing the spreading capacity of mesenchymal stem cells according to PMA treatment (* p <0.05 vs. control).

Figure 3 is a diagram showing the results confirmed by the separation rate of the mesenchymal stem cells according to PMA treatment by trypsinization assay (* p <0.05 vs. control).

Figure 4 shows the spread (a) and adhesion (b) of mesenchymal stem cells after treatment with mesenchymal stem cells with protein kinase C (PKC) activator (PMA) and inhibitor (rotlerin). (A: * p <0.001 vs. control, * p <0.001 vs. PMA alone group b: * p <0.05 vs. control, * p <0.05 vs. PMA alone group Bar: 100 μm).

5 and 6 show the attachment of mesenchymal stem cells following treatment with PKC activator (PMA) and inhibitor (rotlerin) in fibronectin secondary structure (FIG. 5), cardiogel tertiary structure (FIG. 6). (Fig. 5 * p <0.001 vs. DMSO, * * p <0.001 vs. PMA, p <0.005 vs. DMSO and p <0.005 vs. PMA) Fig. 6: ¶ p <0.01 vs. DMSO, ¶¶ p <0.01 vs. PMA, † p <0.05 vs. DMSO and †† p <0.005 vs. PMA Bar: 100 μm)

FIG. 7 shows phosphorylation of FAK, a cell adhesion-associated signaling molecule, paxillin (B), and talin (C) following treatment with PKC activator (PMA) and inhibitor (rotlerin). , Figure showing the results of confirming the expression change of vinculin (D) by immunoassay (* p <0.01 vs. untreated group and * * p <0.05 vs. PMA-treated group).

Figure 8 shows the results of confirming the expression changes of Rac1 following treatment with PKC activator (PMA) and inhibitor (rotlerin) (* p <0.01 vs. untreated group and * * p <0.05 vs. PMA-treated) group).

9 to 12 are diagrams showing the results of confirming the intracellular location of adhesion-related molecules using double immunostaining (vinculin and p-FAK (FIG. 9), integrin α5 and paxillin (FIG. 10), integrin αvβ3 and paxillin (FIG. 11), vinculin and integrin αV (FIG. 12)) (Bar: 50 μm).

Figure 13 is a diagram confirming the location and viability of the mesenchymal stem cells after transplantation into the rat mesenchymal stem cells pre-treated with PMA.

14 is a diagram showing the results of measuring the size change of the myocardial infarction of the heart transplanted with PMA-treated mesenchymal stem cells through TTC staining.

15 is a diagram showing the results of measuring the fiber formation site of the heart implanted with PMA-treated mesenchymal stem cells by Masson's trichrome staining.

Figure 16 shows the change in the development of dead cardiomyocytes showing a TUNEL-positive response following transplantation of PMA-treated mesenchymal stem cells using a TUNEL assay.

The present invention provides a composition for promoting cell adhesion, comprising a protein kinase C (PKC) activator as an active ingredient.

PKC activator according to the present invention, when processed in stem cells, activates PKC, promotes phosphorylation of FAK, involved in cell adhesion-related signal transduction, paxillin, talin, vinculin By promoting the expression of the stem cells can be effectively attached to the site of implanted heart damage.

The PKC activator is not particularly limited in kind, and may be preferably PMA (phorbol myristate acetate) or diacylglycerol.

The PKC activator is preferably included at a concentration of 50 to 500 nM, more preferably may be included at a concentration of 100 nM.

The stem cells may include adult stem cells, embryonic stem cells, mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, cord blood stem cells and dedifferentiated stem cells, preferably mesenchymal stem cells. The mesenchymal stem cells can be used regardless of where they originate from. Mesenchymal stem cells can be obtained from known mesenchymal stem cell sources, for example bone marrow, tissue, embryo, umbilical cord blood, blood or body fluids. Animals to be harvested, such as bone marrow and tissue may be mammals, and when the animal is a human, bone marrow, tissue, etc., are administered as a cell therapy, stem cells having increased adhesion by treatment with the protein kinase C activator of the present invention. It may be the patient's own or someone else's. Methods for obtaining mesenchymal stem cells from such known mesenchymal stem cell sources are well known in the art.

Attachment of the stem cells means that the stem cells to be transplanted are transplanted to a transplantation site that is in need of treatment, preferably a heart injury site. When the attachment of the transplantation site and the transplanted stem cells is promoted, the survival rate of the transplanted stem cells increases. It can effectively treat heart disease.

In another aspect, the present invention provides a method for promoting stem cell adhesion, characterized in that the protein kinase C (hereinafter, PKC) activator is treated to the stem cells.

The method of treating the stem cells with the PKC activator is not particularly limited, and any method may be used as long as the method allows PKC in the stem cells to be activated by contacting the stem cell with the PKC activator for a predetermined time. For example, the treatment of the PKC activator can be performed by culturing stem cells in a medium containing the PKC activator.

The medium may include a medium generally used in the culture of stem cells. Although not limited thereto, such a medium may include, for example, Minimum Essential Medium alpha (MEM-alpha), Mesenchymal Stem Cell Growth Medium (MSCGM), Dulbecco's Modified Eagle's Medium (DMEM), and the like.

The attachment of the stem cells may be characterized in that the cardiac cell damage site, the heart cells include all cells in the process of differentiation into cardiomyocytes or cardiomyocytes.

The present invention also provides a pharmaceutical composition for treating ischemic heart disease comprising a stem cell increased adhesion ability treated with a protein kinase C activator, and the same as an active ingredient.

Stem cells with increased adhesion capacity are characterized by increased phosphorylation of focal adhesion kinase (FAK) and increased expression of Rac1, which is paxillin, tallin, vinculin, and GTase.

The ischemic heart disease may include angina or myocardial infarction.

When transplanted stem cells treated with the protein kinase C activator, increased adhesion ability to the ischemic heart disease site, myocardial infarction site can be reduced and the fibrosis of the heart damage site can be suppressed, effectively inhibiting the death of cardiomyocytes It can effectively treat ischemic heart disease.

The stem cells may be used for transplantation as it is, and may be transplanted as a composition to which various agents are added or genes are introduced to improve the treatment efficiency according to the transplantation.

In the preparation of the composition of the present invention, for example, 1) the addition of a substance that enhances the cell proliferation rate of the present invention or promotes further differentiation into cardiac cells, or the introduction of a gene having such an effect, 2) the present invention The addition of a substance that improves the survival rate in the damaged area of cells, or the introduction of a gene having such an effect, 3) the addition of a substance that prevents the adverse effects of the cells of the present invention from damaged tissues, or such effects. Introduction of a gene having the effect, 4) addition of a substance that extends the life of a donor cell, or introduction of a gene having such an effect, 5) addition of a substance that modulates the cell cycle, or a gene having such an effect 6) the addition of a substance aimed at suppressing the immune response, or the introduction of a gene with such an effect, 7) the activation of energy metabolism The addition of a substance or introduction of a gene having such an effect, 8) a substance that enhances the ability of the donor cells in the host tissue, or an introduction of a gene having such an effect, 9) a substance that improves blood flow, or Introduction of genes having such an effect (including angiogenesis induction), but is not limited thereto.

The method of administering stem cells of the present invention is not particularly limited, and for example, topical administration, intravenous administration, intraarterial administration, and the like can be used.

Specifically, cell transplantation into a patient can be administered by any route as long as it can induce migration to the disease site. For example, the cells to be transplanted may be collected by collecting them in a syringe in a suspended state using physiological saline, etc., exposing tissue damaged by surgery, and injecting directly into the damaged area with a needle. Consideration may be given to loading into a vehicle having a means for directing the lesion. Thus, the activated stem cells of the invention may be topical (including buccal, sublingual, skin and intraocular administration), parenteral (including subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular and cerebrospinal fluid) or Administration can be via several routes including percutaneous administration, preferably parenteral administration, most preferably direct to the affected area.

The pharmaceutical composition of the present invention can be used as a cell therapeutic agent, and the cell therapeutic agent is a drug used for the purpose of treatment, diagnosis, and prevention by cells and tissues prepared through isolation, culture, and special manipulation from humans (US FDA regulation). For the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating and screening live autologous, allogeneic, or heterologous cells in vitro or otherwise altering the biological properties of the cells to restore the function of the cells or tissues. Means the medicine used.

In addition, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient and diluent in addition to the active ingredient described above for administration. For example, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil.

The pharmaceutical compositions of the present invention may be prepared in various parenteral or oral administration forms according to known methods. As representative of formulations for parenteral administration, isotonic aqueous solutions or suspensions are preferred for injectable formulations. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer.

The effective dosage of the pharmaceutical composition of the present invention may vary depending on the age and gender of the patient, but may be administered to the subject by suspending in a suitable diluent at a concentration of 4x10 ⁵ to 4x10 ¹⁰ cells / ml, preferably 4x10 ⁷ cells. It can be used by suspending at a concentration of / ml. The diluent is used for the purpose of protecting and maintaining the cells and facilitating their use when injected into the desired tissue. The diluent may be a saline solution, a phosphate buffer solution, a buffer solution such as HBSS, plasma or blood components.

The composition of the present invention can be used alone or in combination with methods using surgery, chemical therapy, radiotherapy, hormone therapy, drug therapy and biological response modifiers for the treatment of ischemic heart disease.

Hereinafter, the present invention will be described in detail by production examples and examples. However, the following Preparation Examples and Examples are merely to illustrate the present invention, and the content of the present invention is not limited by the following Preparation Examples and Examples.

The following data are expressed as means ± SEM and the significant difference between the two groups was measured by Student's t-test. Two or more groups were used for comparison and one-way ANOVA was performed using Bonferroni's correction. P value must be less than 0.05 to be significant.

Example 1. Isolation and Culture of Mesenchymal Stem Cells

Mesenchymal stem cells were collected by extracting bone marrow-derived mesenchymal stem cells from the femur and tibia of a 4 week old male Sprague-Dawley rat (about 100 g). The obtained mesenchymal stem cells were cultured in 10 ml of medium consisting of DMEM (Dulbecco's modified Eagle's medium) to which 10% fetal bovine serum, 1% penicillin and streptomycin solution were added. The bone marrow isolated from the upper layer of 1.073 g / ml percoll was added and centrifuged to wash the mononuclear cells obtained at the interface twice, and then mixed with 10% FBS-DMEM and dispensed into the flask at 1 × 10 ⁶ cells / 100 cm ² . . Incubation was in a Forma Scientific incubator with 37 ° C., 95% air, 5% CO ₂ . After 48 to 72 hours, nonadsorbent cells were removed and adsorbent cells were washed twice with PBS. Fresh medium was added and cultured with changing medium every 3-4 days for about 10 days. In order to obtain only mesenchymal stem cells, immunophenotyping was performed using various markers on the cell surface and cells. After obtaining the cells, the cells were washed with PBS and labeled with the FITC-attached antibodies (CD14, CD34, CD71, CD90, CD105, CD106, ICAM-1). Goat anti-mouse IgG (goat anti-mouse IgG) with FITC was used as a secondary antibody. Labeled cells were identified using a flow cytometer, and the results were analyzed using Cell Quest Pro Software. The mesenchymal stem cells obtained were used for in vivo and in vitro experiments in passage 2 and passage 3 cells.

Example 2. Mesenchymal Stem Cell Attachment and Spread Analysis

After the mesenchymal stem cells were suspended, 2 x 10 ⁴ cells were added to each well in a 6 well plate. The cells were allowed to adhere to the plate at 37 ° C. and 5% CO ₂ for 1 hour. Subsequently, phorbol 12-myristate 13-acetate (PMA), a protein kinase C (hereinafter, PKC) activator, was treated for 2 to 8 hours, and the attachment of mesenchymal stem cells was determined by an adhesion assay and spread analysis. It was confirmed using (spreading assay). At this time, DMSO was treated to mesenchymal stem cells as a control.

2.1 Increased adhesion of mesenchymal stem cells by PMA treatment

To confirm the attachment of mesenchymal stem cells, the mesenchymal stem cells were treated with PMA (20, 50, 100, 200, 500 nM) at various concentrations, and then carefully washed the mesenchymal stem cells with PBS three times. Four different parts were taken using a phase contrast microscope. All experiments were performed using three different wells and repeated three times.

The results are shown in FIG.

As shown in FIG. 1, 4 hours after PMA treatment, a difference in adhesion ability was observed between DMSO and PMA treatment groups, and mesenchymal stem cells showed the highest increase in PMA 100 nM concentration ( a). In the case of treatment with 100 nM concentration of PMA, it was confirmed that adhesion capacity increased 4.4 times in 4 hours (b). In subsequent experiments, all PMAs were treated with 100 nM.

2.2 Increased spread of mesenchymal stem cells by PMA treatment

In order to analyze the spreading capacity of stem cells, the cells were incubated at 37 ° C. and 5% CO ₂ conditions in 4 wells for 4 hours, washed three times with PBS, and fixed with 3% formaldehyde (formaldehyde). Fixed cells were stained with coomassie blue and residual material was removed. Finally, four parts were photographed with a phase contrast microscope.

The results are shown in FIG.

As shown in Figure 2, mesenchymal stem cells treated with PKC promoter, PMA, at the same concentration, increased the ability to spread by 4.5 times compared to the control.

2.3 Trypsinization assay of mesenchymal stem cells by PMA treatment

In order to confirm the effect of the PKC activator PMA on the adhesion of stem cells, the mesenchymal stem cells were cultured for one day, and then treated with PMA for 4 hours and a trypsinization assay was performed. Cells were plated about 90% and trypsinized at 37 ° C. for 3 minutes. The fallen cells were washed away, and the cells attached to the plates were incubated at 37 ° C. for 1 hour, and then nine cells were counted per plate.

The results are shown in FIG.

As shown in Figure 3, mesenchymal stem cells treated with PMA significantly reduced the separation rate compared to the control.

Example 3. Adhesion of mesenchymal stem cells by PKC activator and inhibitor

In order to confirm whether the increase of mesenchymal stem cell adhesion and spreading ability by PMA treatment is influenced by the promotion of PKC activity, mesenchymal stem cells were treated with PMA, a PKC activator, and rottlerin, a PKC activity inhibitor, The adhesion and spreading ability of stem cells were compared with the control.

The results are shown in FIG.

As shown in FIG. 4, the adhesion of mesenchymal stem cells treated with PMA, a PKC activator, was increased, and the adhesion with rottlerin, an activity inhibitor, decreased. In addition, in the experimental group treated with PMA and rottlerin at the same time confirmed the adhesion capacity similar to the control group (a). In addition, as a result of measuring the separation ratio of the cells, the experimental group treated with PMA showed a separation ratio of about 25% compared to the control group (b). Through these results, it was confirmed that the treatment of PKC activator to mesenchymal stem cells can increase the adhesion of stem cells.

Example 4 Changes in the Adhesion of Stem Cells in Cardiogel

Fibroblasts and Vitronectin are components of the extracellular matrix and are involved in cell growth and differentiation. Therefore, in order to confirm whether the PKC activator can play a functional role in the environment after in vivo culture, an experiment using a substrate having a tertiary structure was performed using a cardiogel. Cardiogel, a substrate of tertiary structure derived from cardiac fibroblasts, used a method modified from the method of Cukierman ³⁹ . Cardiogel simulates the cardiomyocyte environment using fibroblasts from the heart. Cardiogels were dispensed at 2 × 10 ⁵ in a 35 mm dish and the medium was changed every 48 hours until tissue deprived of cell characteristics. After washing with PBS, 1 ml of extraction buffer in 0.5% Triton X-100, 20 mM NH ₄ OH was added to warm PBS, and cell lysis was observed using an inverted microscope until no intact cells were seen. In order to wash the cell wall debris with PBS and to minimize DNA debris in the tissue 1ml DNase (10 unit DNase / 1 ml PBS) was added and incubated at 37 ℃ for 30 minutes. Prior to use, 3 ml of PBS containing 100 U / ml penicillin, 100 μg / ml streptomycin, 0.25 μg / ml Fungizone was added to tissue coated plates and refrigerated until use. Since cardiogel is a tertiary structure, it may be different from the adhesion ability in the secondary structure. Therefore, in order to confirm the adhesion ability of mesenchymal stem cells by PMA treatment, which is a PKC activator in a three-dimensional cardiogel and a fibronectin matrix of secondary structure, the mesenchymal stem cells were cultured on a fibronectin-coated plate and treated with PMA. Then incubated for 2 and 16 hours or in cardiogel for 2 and 16 hours. After incubation, the appearance of the cells was observed under a control microscope.

The results are shown in FIGS. 5 and 6.

As shown in Figure 5 and 6, the treatment of PMA in both secondary and tertiary structure significantly increased the adhesion of stem cells. In addition, the experimental group treated with Rotlin, a PKC inhibitor, showed a marked decrease in cell adhesion, and the experimental group treated with PMA and Rotlin at the same time showed a similar degree of adhesion as the control group. It also showed higher adhesion in three-dimensional cardiogels (FIG. 6) than in fibronectin-covered plates (FIG. 5).

Example 5 Changes in Cell Attachment Associated Signaling by PKC Activator Treatment

By PMA treatment that induces the activation of PKC activity, it was confirmed by immunoassay whether the expression of adhesion-associated signal transduction molecules was changed in mesenchymal stem cells. After washing the mesenchymal stem cells with PBS, 20mM Tris (pH7.5), 150mM NaCl, 1mM Na ₂ -EDTA, 1m MEGTA, 1% Triton, 2.5mM sodium pyrophosphate, 1mM ßß-glycerophosphate (glycerophosphate), 1 mM Na ₃ VO _4, 1 mg / ml leupeptin and 1 mM PMSF (phenylmethylsulfonylfluoride) were dissolved in cell lysis buffer (Cell Signaling, Beverly, MA, USA). Protein concentration was measured using a Bradford Protein assay kit (Bio-Rad Laboratories, Richmond, CA). The same amount of protein from the whole cell lysate was lowered on 12% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. 5% fat-free dry milk was added to Tris-buffered saline-Tween 20 (TBS-T, 0.1% Tween20) to block the membrane at room temperature for one hour. The membrane was washed twice with TBS-T and then the primary antibody was reacted at room temperature for 1 hour or overnight at 4 ° C. After this process, the mixture was wiped with TBS-T three times for 10 minutes, and incubated for 45 minutes by adding a secondary antibody bound to horseradish peroxidase (HRP) at room temperature. After performing the washing process once again, the band was confirmed by ECL reagent (enhanced chemiluminescence reagent, Santa Cruz Biotechnology). The intensity of the band was measured using a photo-image system (molecular dynamics, Sunnyvale, CA).

The results are shown in FIG.

As shown in FIG. 7, the phosphorylation of focal adhesion kinase (FAK), a signaling molecule associated with cell adhesion, was increased by 28.5% by treatment of PMA, a PKC activator (A), paxillin (B), The expressions of talin (C) and vinculin (D) were also markedly increased by 46.2%, 33% and 20%, respectively. This increase in expression was reduced by treatment with Rotlin, a PKC activity inhibitor. However, when the PKC activator and the inhibitor were treated simultaneously, the phosphorylation of FAK increased by 50%, and the expression of paxillin, thallin, and vinculin increased by 50%, 45%, and 47.5%, respectively, compared to the PKC activity inhibitor alone group. Confirmed.

Example 6 Changes in Expression of Small GTPases by PKC Activator Treatment

The activity of Rho GTPases can be achieved by measuring the expression of Rac1. At the cell attachment site, PKC affects the actin cytoskeleton through its effects on Rho GTPases, and in cell spreading, Rac1 is located under the mechanism of PI3-kinase and PKC, which include the function of the integrin cytoplasmic domain. The argument to be placed. Therefore, it was confirmed that the expression of PMA, a PKC activator, affects the signal transduction process of cell spreading through its expression.

The results are shown in FIG.

As shown in FIG. 8, expression of Rac1 was doubled by treatment with PMA. In addition, when simultaneously treated with PKC activator and inhibitor, the expression level of Rac1 remained similar to that of the control. Through this, it was confirmed that when the PKC activator PMA is treated to the stem cells, PKC is activated and the lower signaling process is activated to increase the adhesion and spreading ability of the stem cells.

Example 7 Expression of Cell Attachment Factors Using Immunocytochemistry

Double immunostaining was used to determine whether the adhesion-related molecules were in the same position in the cell. Cells were grown in 4-well plastic petri dishes and washed twice with PBS. Then, 0.5 ml of 4% paraformaldehyde diluted in PBS was added thereto and reacted for 30 minutes. The cells were washed with PBS again and the cells were permeated with PBS containing 0.2% Triton X-100. Integrin α5, integrin αvβ3, integrin αV, p-FAK, paxillin and vinculline primary antibodies were added to cells blocked with PBS containing 10% goat serum. The cells were washed with PBS three times for 10 minutes, followed by attaching secondary goat anti-rabbit IgG with fluorescein isothiocyanate (FITC) and goat anti-mouse IgG with Texas red for 1 hour. All photographs were identified by light fluorescence microscopy (LSM 700, Carl Zeiss. Thomwood, NY) using an excitation filter.

The results are shown in FIGS. 9 to 12.

As shown in Figs. 9-12, vinculin and p-FAK (Fig. 9), integrin α5 and paxillin (Fig. 10), integrin αvβ3 and paxillin (Fig. 11) and vinculin by treatment with PKC modulators. It was confirmed that the strength of the overlap between and integrin αV (FIG. 12) was increased by treatment with PMA, an inhibitor of PKC activity. The intensity of overlap signal was lower in the stem cells treated with PKC inhibitor, Rotlerin, compared to the control group, and similar to the control group in the stem cells treated with PKC activator and inhibitor at the same time.

Example 8. Regeneration Effect of Injured Myocardium by Transplantation of PMA Treated Mesenchymal Stem Cells

By improving the adhesion and spreading ability of PMA-treated mesenchymal stem cells, it was confirmed using myocardial infarction model rats to increase the regeneration effect of myocardium when transplanted treated mesenchymal stem cells.

8.1 Induction of Myocardial Infarction in Rats

To induce myocardial infarction in rats, 8-week-old Sprague-Dawley male rats (approximately 205 g) were subjected to positive pressure circulation (180 ml / min) after airway intubation under normal anesthesia, and the circulation was Harvard ventilator. Was maintained in room air to which oxygen (2 L / min) was added. After exposing the rat heart to left thoracotomy, the left anterior descending paper was ligated with 6-0 silk suture and left for 1 hour. Before performing the experiment, mesenchymal stem cells were treated with PKC activator or activity inhibitor for 4 hours. Before heart closure of rats, mesenchymal stem cells labeled with DAPI (4 ', 6'-diamidine-2'-phenylindole dihydrochloride) were injected into the heart. Thereafter, the ligation was released to induce reperfusion, the wound was closed with a suture, the ventilator was removed, and recovered from the cage to create a myocardial infarction model.

8.2 Transplantation and Labeling of Mesenchymal Stem Cells

Mesenchymal stem cells were labeled with DAPI and the sterile DAPI solution was transplanted on the same day as the cell transplanted for 30 minutes until the final concentration was 50 μg / ml. Cells were washed 6 times with PBS and cells without DAPI were removed. Cells labeled with DAPI were detached with 0.25% trypsin, and cells to be used for transplantation were suspended in medium without serum. After inducing myocardial infarction, cells suspended in 1 × 10 ⁶ mesenchymal stem cells in 100 μl serum-free medium were transplanted using a 30 gauge needle. Cells were injected into the front and side of the interface without infarction. In this experiment, four experimental groups (normal rats, rats not infused after myocardial infarction, rats inoculated with mesenchymal stem cells after induction of myocardial infarction, and mesenchymal stem cells activated with PKC after induction of myocardial infarction) Rats implanted) were used. One experimental group injected mesenchymal stem cells stained with DAPI and measured the survival of mesenchymal stem cells after 3 days. And then, the mesenchymal stem cells were transplanted into two experimental groups and used for analyzing the cell shape after one week. The other experimental groups were used for cardiac function evaluation after three weeks.

Transplantation results are shown in FIG. 13.

As shown in FIG. 13, mesenchymal stem cells pretreated with PMA adhered to the border region of the infarct and the normal portion, and most of the mesenchymal stem cells were attached to the infarct border region of the left ventricle as compared with the control group. In addition, the number of DAPI-labeled cells was confirmed to be higher than that of the PMA pretreatment group, which indicates that mesenchymal stem cells can be effectively attached by PMA treatment.

8.3 Histological Analysis of Cell Transplanted Heart

Seven days after transplanting the mesenchymal stem cells, the animals were sacrificed and the heart was extracted. The heart was perfused with 10% (v / v) neutral buffered formaldehyde for 24 hours, fixed, cut horizontally, and soaked in paraffin as usual. The slices cut to a thickness of 5 μm were coated on gelatin-coated glass slides so that staining could work well on the continuous sections of the tissue at the implant site.

8.3.1 Identifying the size of myocardial infarction

TTC (2,3,5-Triphenyltetrazolium chloride) staining was used to measure cardiac tissue viability and myocardial infarction size. The tissues were cut and immersed in 1% TTC (Sigma, St. Louis, MO) solution at pH 7.4 and placed at 37 ° C. for 20 minutes. Tissues were placed in 10% formalin and left overnight at 4 ° C. The heart was cut transversely and the percent infarct size was calculated at what percentage of the total left ventricle. Both tissues stained with TTC were photographed with a digital camera.

The results are shown in FIG.

As shown in FIG. 14, the model of the mesenchymal stem cells pretreated with PMA significantly reduced the size of myocardial infarction compared with the untreated control.

8.3.2 Confirm Fibrosis of the Heart

Fibrosis was analyzed using Masson's trichrome staining method. Fibrotic sites of the group injected with PMA-treated mesenchymal stem cells were measured using metamophor, and the expression rate was measured against the total left ventricle.

The results are shown in FIG.

As shown in FIG. 15, fiber formation in the myocardial infarction site was reduced by about 15% in the PMA pretreated group. Through this, it was confirmed that the stem cells pretreated with PMA, a PKC activator, can more effectively improve the fibrosis of myocardial infarction.

8.3.3 Confirmation of Death of Heart Cells

TUNEL assay (Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay) was performed according to the manufacturer's method (Chemicon International, Temecula, CA). Paraffin of 5 μm thick tissue was removed and rehydrated and washed with PBS. The sections were then treated with 3.0% H ₂ O _{2 and} then treated with terminal deoxynucleotidyl transferase (TDT) enzymes at 37 ° C. for 1 hour. Subsequently, nucleotides with digoxigenin were reacted at 37 ° C. for 30 minutes. 3,3-DAB (3,3-diamino benzidine Vector Laboratories, Burlingame, Calif.) Was treated for 5 minutes. Finally, the sections were counterstained with methyl green, covered with coverslip, and observed under an optical microscope. Each group made 5 sections and observed at 5 different sites.

The results are shown in FIG.

As shown in FIG. 16, when the stem cells treated with PMA, a PKC activator, were transplanted, the generation of dead cardiomyocytes showing TUNEL-positive responses was reduced by about 20% in the PMA treated group compared to the control group. Through this, it can be seen that transplantation of stem cells treated with a PKC activator can inhibit the death of cardiomyocytes.

8.4 Measurement of Cardiac Function Using Left Ventricular Catheter Insertion

Left ventricular catheterization was performed after infarction. While the animals were anesthetized, a Millar Mikro-tip 2F pressure transducer (model SPR-838, Millar instruments, Houston, Texas) was inserted through the right carotid artery into the left ventricle. The pressure-volume ring was stored in real time and all data were analyzed offline using PVAN3.5. The experiment calculated the average after 3 trials. Relative volume units were normalized to heparinized blood using graduated cuvettes.

The results are shown in Table 1.

Table 1

As shown in Table 1, the function of the heart in rats implanted with PMA treated mesenchymal stem cells calculated using the pressure-volume ring was shown to gradually improve after myocardial infarction. In particular, the ejection fraction (EF), stroke volume (SV), cardiac output (cardiac output) was significantly increased in the group transplanted with PMA pretreated mesenchymal stem cells compared to the control group. In addition, PMA-treated mesenchymal stem cell transplanted rats had increased LV function.

Claims (17)

1. Comprising a protein kinase C (activator) activator as an active ingredient, composition for promoting stem cell adhesion.
2. The composition of claim 1, wherein the protein kinase C activator is PMA (phorbol myristate acetate) or diacylglycerol.
3. According to claim 1, wherein the protein kinase C activator, characterized in that it comprises 50 to 500nM concentration, composition for promoting stem cell adhesion.
4. The method of claim 1, wherein the stem cells are adult stem cells, embryonic stem cells, mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, umbilical cord blood cells and dedifferentiated stem cells, characterized in that at least one selected from the group consisting of stem cells , Stem cell adhesion promoting composition.
5. The composition of claim 4, wherein the mesenchymal stem cells are obtained from one or more selected from the group consisting of bone marrow, tissue, embryo, umbilical cord blood, blood, and body fluids.
6. A method for promoting adhesion of stem cells, characterized in that the stem cells are treated with a protein kinase C activator.
7. The method of claim 6, wherein the protein kinase C activator is PMA (phorbol myristate acetate) or diacylglycerol.
8. The method of claim 6, wherein the attachment of the stem cells is performed at the site of cardiac cell damage.
9. Stem cells with increased adhesion, treated with protein kinase C activators.
10. The stem cell of claim 9, wherein the stem cell has increased phosphorylation of focal adhesion kinase (FAK).
11. The stem cell of claim 9, wherein the stem cells have increased expression of paxillin, talin, and vinculin.
12. 10. The stem cell of claim 9, wherein the stem cell has an increased expression of Rac1.
13. A protein composition for treating ischemic heart disease, comprising a stem cell of increased adhesion, treated with a protein kinase C activator, as an active ingredient.
14. The pharmaceutical composition for treating ischemic heart disease of claim 13, wherein the ischemic heart disease is angina pectoris or myocardial infarction.
15. The pharmaceutical composition for treating ischemic heart disease according to claim 13, wherein the stem cells reduce the myocardial infarction site when transplanted into the heart cells.
16. The pharmaceutical composition for treating ischemic heart disease according to claim 13, wherein the stem cells inhibit fibrosis of the heart injury site.
17. The pharmaceutical composition for treating ischemic heart disease of claim 13, wherein the stem cells inhibit the death of cardiomyocytes.

Images