WO2017197648A1 - Pharmaceutical composition for treating cardiopathy - Google Patents

Abstract

A pharmaceutical composition for treating diabetic cardiopathy. The composition comprises an ester type catechin (EGCG) and adipose-derived stem cells. The ester type catechin (EGCG) could enhance the ability of the adipose-derived stem cells and increase the ability of the adipose-derived stem cells in repairing damaged tissues.

Description

Medicine composition for treating heart disease Technical field

The present invention provides a pharmaceutical composition for repairing diabetic heart disease and a method of using the same, wherein the pharmaceutical composition for repairing diabetic heart disease comprises adipose stem cells pretreated with ester catechin (EGCG). The ability of the green tea catechin to pretreat adipose stem cells to repair damaged tissues has a significant increase.

Background technique

Diabetes Mellitus (DM) is a disorder of sugar, protein and fat metabolism caused by the lack of insulin in the body or the normal physiological action of insulin in target cells. It is clinically proven that many diseases are accompanied by diabetes. And, these diseases are called complications of diabetes. Complications of diabetes include cardiovascular disease, kidney disease, peripheral vascular disease, eye disease, liver disease or neuropathy or peripheral neuropathy. Among them, 2 out of every 4 diabetic patients have abnormal cardiac function, indicating that diabetic heart disease is a major complication of diabetic patients.

Related studies have pointed out that the damage caused by diabetes to the heart is through the blood sugar itself or advanced glycation end products (AGEs). Regardless of the source of the stimulus, the oxidative stress of the cardiomyocytes rises and rises. Oxidative stress destroys the glandular glands in cardiomyocytes, and the expression levels of apoptosis-related proteins (such as caspase-3 and t-Bad) increase. In contrast, the expression level of cell surviving proteins such as p-Akt is decreased, and further causes pathological reactions of cardiomyocytes, such as apoptosis, cardiomyocyte hypertrophy, inflammatory reaction and even fibrotic reaction. The manifestation of these pathological reactions will eventually lead to a decline in cardiac function, and after the myocardial inflammation occurs, the damaged cells cannot Self-regeneration, the current medical treatment can not make the damaged cells self-regeneration, so it can not restore the heart function and solve the problem of hyperglycemia.

Stem cell therapy has the ability to treat heart and cardiovascular lesions caused by diabetes, allowing damaged cells to self-regenerate and restore heart function. However, studies have found that stem cells have poor regenerative capacity at high sugar concentrations. How to maintain stem cells in a high-glucose environment can be an urgent problem for autologous stem cells to repair diabetic heart disease.

Summary of the invention

The present invention provides a pharmaceutical composition for treating heart disease, wherein the pharmaceutical composition comprises stem cells.

Preferably, the heart disease is a heart disease caused by diabetes or hyperglycemia.

Preferably, the pharmaceutical composition further comprises ester catechin (EGGG).

Preferably, the stem cells are adipose stem cells.

Preferably, the adipose stem cells are pretreated with ester catechin for 2 hours.

Preferably, the concentration of the ester catechin of the pretreated adipose stem cells is less than 20 μM.

Preferably, the concentration of the ester catechin of the pretreated adipose stem cells is 5-15 μg/mL.

The present invention provides a method of preparing a pharmaceutical composition for treating heart disease, comprising 1 × 10 ⁵ adipose stem cells treated with ester catechin.

Preferably, the heart disease is caused by diabetes or hyperglycemia.

The present invention provides a method for treating heart disease comprising administering 1 × 10 ⁵ stem cells to a body by intravenous injection.

Preferably, the heart disease is a heart disease caused by diabetes or hyperglycemia.

Preferably, the pharmaceutical composition further comprises an ester catechin.

Preferably, the stem cells are adipose stem cells.

Preferably, the adipose stem cells are pretreated with ester catechin for 2 hours.

Preferably, the concentration of the ester catechin of the pretreated adipose stem cells is less than 20 μg/mL.

Preferably, the concentration of the ester catechins of the pretreated adipose stem cells is 5-15 μM.

The present invention provides a method for increasing the tolerance and mobility of stem cells to sugars, the method comprising adding an ester catechin to a culture medium of stem cells.

Preferably, the ester catechin has a concentration of 5-15 μM.

Preferably, the method further comprises removing the stem cells in a medium containing the stem cells of the ester catechin for 2 hours.

This Summary is not an extensive overview of the invention, and is not intended to be an The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, can be readily understood by those of ordinary skill in the art.

DRAWINGS

Figure 1 shows the characteristics of adipose stem cells, distinguishing the positive label and negative label of the second generation of adipose stem cells;

Figure 2 shows the results of the differentiation ability test of the second generation of adipose stem cells;

Figure 3 is a diagram showing the distribution of colony of adipose-derived stem cells by the ability of green tea EGCG to pass through CXCR4 in a medium with high glucose concentration (33 mM);

Figure 4 is a graph showing the ability of green tea EGCG to enhance the ability of adipose stem cells and the ability of adipose stem cells (ADSC) to move through CXCR4 in a medium with high glucose concentration (33 mM);

Figure 5 is a Western blot analysis of pretreatment of adipose-derived stem cells with different doses of green tea EGCG. (ADSC) protein expression level results;

Figure 6 is a Western blot method, the results of protein expression of green tea EGCG pretreatment and adipose stem cells (ADSC) without green tea EGCG pretreatment in the presence of CXCR4 siRNA;

Figure 7 shows the mobility test results of adipose-derived stem cells (ADSC) with the ability to move green tea EGCG pretreatment and adipose stem cells (ADSC) without green tea EGCG pretreatment, with or without CXCR4 siRNA;

8 is a result of protein expression of cardiomyocyte H9c2 co-cultured by Western blotting method, adipose stem cells (ADSC), and adipose-derived stem cells (ADSC) pretreated with green tea EGCG (10 μM);

Figure 9 is a graph showing the protein expression level of cardiomyocyte H9c2 co-cultured with stem cells (ADSC) and green tea EGCG (10 μM) pretreated adipose-derived stem cells (ADSC) in a high glucose (33 mM) environment;

Figure 10 is a graph showing blood glucose levels and body weight results of diabetic mice in autologous transplantation of stem cells (ADSC) and adipose-derived stem cells (ADSC) pretreated with green tea EGCG;

Figure 11 is a cardiac ultrasound map of diabetic mice in autologous transplantation of stem cells (ADSC) and adipose-derived stem cells (ADSC) pretreated with green tea EGCG;

Figure 12 is a (A) cardiac blood ejection rate (EF%) and (B) cardiac compression rate (FS%) of diabetic mice in autologous transplantation of stem cells (ADSC) with green tea EGCG pretreated with stem cells (ADSC);

Figure 13 is a diagram showing the cardiac tissue changes of diabetic mice in autologous transplantation of stem cells (ADSC) and adipose-derived stem cells (ADSC) pretreated with green tea EGCG;

Figure 14 is a graph showing the results of analysis of survivin protein index of diabetic rat in autologous transplantation of stem cells (ADSC) and adipose-derived stem cells (ADSC) pretreated with green tea EGCG;

Figure 15 is a Western blot analysis of protein expression related to myocardial apoptosis in diabetic mice with autologous transplantation of stem cells (ADSC) and green tea EGCG pretreated adipose-derived stem cells (ADSC);

Figure 16 is a quantitative analysis of apoptotic signals of diabetic mice in autologous transplantation of stem cells (ADSC) and adipose stem cells (ADSC) pretreated with green tea EGCG;

Figure 17 is a graph showing the longevity protein of the heart of diabetic mice in autologous transplantation of stem cells (ADSC) and adipose-derived stem cells (ADSC) pretreated with green tea EGCG;

Figure 18 shows the cardiac ultrasonography and ventricular hypertrophy protein in diabetic rats with autologous stem cells (ADSC) and adipose-derived stem cells (ADSC) pretreated with green tea EGCG. (A) is to observe the end-diastolic thickness of the ventricular septum ( Interventricular septal thickness at end diastole (IVSd), (B) for the observation of ventricular septal thickness at end systole (IVSs);

Figure 19 shows the cardiac ultrasonography and ventricular hypertrophy protein in diabetic mice with autologous stem cells (ADSC) and adipose-derived stem cells (ADSC) pretreated with green tea EGCG. (A) is to observe the left ventricular posterior wall end-diastolic thickness ( Left ventricular posterior wall thickness at end diastole (LVPWd), (B) is the left ventricular posterior wall thickness at end systole (LVPWs) results;

Figure 20 is a Western blot diagram of hypertrophic and ventricular hypertrophy protein in hypertrophic-associated protein analysis of myocardial tissue in diabetic mice with autologous transplantation of stem cells (ADSC), adipose-derived stem cells (ADSC) pretreated with green tea EGCG;

Figure 21 shows the cardiac fibrin index of diabetic rats in autologous transplantation of stem cells (ADSC) and adipose-derived stem cells (ADSC) pretreated with green tea EGCG. The cardiac tissue of each group was Masson. Trichrome section staining results;

Figure 22 is a diagram showing the western fibrin map of diabetic rats in the autologous transplantation of stem cells (ADSC), adipose-derived stem cells (ADSC) pretreated with green tea EGCG, and the homogenization of cardiac tissue.

Figure 23 is a summary of the ability of green tea EGCG to increase the expression of CXCR4 and thereby increase the ability of adipose stem cells to regenerate cardiomyocytes;

detailed description

The description of the embodiments of the present invention is intended to be illustrative and not restrictive The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

The present invention provides a pharmaceutical composition for treating heart disease, which comprises stem cells pretreated with green tea polyphenol, and has the effect of improving stem cell differentiation ability.

The following examples are illustrative and are not intended to limit the scope of the invention.

Example 1: Rat Adipose Stem Cell Extraction and Experiment

Fat stem cell extraction The abdominal fat in the 8-month-old Wistar strain male rats was surgically removed, and the fat was cut into appropriate size. The fat was washed with physiological saline containing antibiotics, and the washed adipose tissue was placed into the second type. The collagenase (0.01%) in physiological saline was heated and stirred in a 37 ° C water bath for about 1 hour, and centrifuged at 3000 rpm for about 10 minutes at room temperature, and the lower layer precipitate was taken out and then cultured in a cell culture dish.

1. Adipose stem cell identification experiment

After the second generation of adipose-derived stem cells are cultured, they are returned to the mice via the tail vein for autologous adipose-derived stem cell transplantation. Before self-transplantation of stem cells, it is necessary to make a review of the cultured adipose-derived stem cells. Determine the cells to be transplanted as stem cells. There are two methods for identifying stem cells in this experiment. One is to identify the positive marker and the negative marker on the adipose stem cell membrane. The positive marker is also the marker that the stem cell must have; otherwise, negative The marker is a marker that stem cells cannot have. It can be seen from Fig. 1 that the expression levels of positive markers CD90 and CD29 on stem cells are 95% and 98%, respectively, whereas the expression levels of negative markers CD45 and CD31 are 0.5% and 0.5%, respectively. In addition to the positive and negative markers on the stem cell membrane, it has to be demonstrated that stem cells must have the ability to differentiate into other types of cells. As shown in Fig. 2, in the test of differentiation ability, stem cells have the ability to differentiate into adipocytes.

2. Gene and siRNA transfer

The cells were cultured in DMEM (dulbecco's modified eagle medium) culture medium, and the cells were transfected with 80% full siRNA, target plasmid (target vector) and DharmaFECT Duo transfection reagent (Dharmacon, Inc.). experiment. Mix 3.5l plasmid (2g/l) and 35l siRNA (20M) in 700l serum-free DMEM (serum-free DMEM) medium (A tube); and DharmaFECT Duo reagent (complex reagent) at 1: The 50 ratio was mixed with serum-free DMEM (serum-free DMEM medium) medium for 5 minutes (B tube). The A and B tubes were then mixed and placed for 20 minutes. Equally add the above mixture of tube A and tube B in a petri dish containing cells, transfer in a 37 ° C incubator, and finally collect the cells for relevant experimental analysis.

3. Determination of protein concentration

The protein was quantified using the Bradford protein assay, which was based on the principle that the protein forms a blue complex with Coomassie billiant blue G-250 (Coomassie Brilliant Blue G250), and the darker the blue, the higher the protein content. The test method firstly adds a one-fifth volume of Bradford protein dye (Bradford protein stain) to a series of known concentrations of BSA (bovine serum albumin), and uses a brightness curve of 595 nm visible light as a standard curve, and then measured in the same way. The OD value of the sample can be used to determine the concentration of the sample protein based on the standard curve.

4. Western ink point method

After the cells were treated with the drug, the culture solution was removed and washed with PBS buffer (phosphate buffer) (3 times), and the cells were scraped from the culture dish using 1 ml of PBS (phosphate buffer) and placed in a centrifuge tube at 4 ° C. Centrifuge at 12,000 rpm for 10 minutes to remove the supernatant, add lysis buffer (lysis buffer, 50 mM Tris pH 7.5, 0.5 M NaCl, 1.0 mM EDTA pH 7.5, 1 mM BME, 1% NP40, 10% glycerol (glycerol), protease inhibitor cocktail Table (protease inhibitor blending agent)) The cells were mixed and placed on ice, shaken once every 5 minutes for 30 minutes, centrifuged at 12,000 g for 10 minutes at 4 ° C, and the supernatant was placed in a new centrifuge tube to measure the protein concentration. Cytoplasmic cytochrome c (cytochrome C) extraction: After cell dosing, the culture solution was removed and washed with PBS buffer (3 times). The cells were scraped from the culture dish using 1 ml PBS and placed in a centrifuge tube at 4 ° C. Centrifuge at 12,000 g for 10 minutes to remove the supernatant, and add extraction buffer (extraction buffer, 50 mM Tris pH 7.5, 0.5 M NaCl, 1.0 mM EDTA pH 7.5, 10% glycerol (glycerol), protease inhibitor cocktail table). With complete mixing, the cells were placed in a grinding tube along with an extraction buffer (50 mM Tris pH 7.5, 0.5 M NaCl, 1.0 mM EDTA pH 7.5, 10 glycerol (glycerol), protease inhibitor cocktail table) on ice. Grinding was carried out, then the homogenate was placed in a new centrifuge tube, centrifuged at 12,000 rpm for 10 minutes at 4 ° C, and the supernatant was placed in a new centrifuge tube to measure the protein concentration. Take 40g of sample protein and add PBS solution (PBS solution) and 5X loading dye (dye) to mix and boil for 10 minutes, then perform SDS-polyacrylamide gel electrophoresis analysis. The SDS-polyacrylamide gel electrophoresis upper layer colloid was 3.75% Stacking gel, and the lower layer colloid was 5% and 12% Separating gel. Fix the prepared plate glue to the electrophoresis device, fill the electrophoresis buffer (Electrode buffer) with the electrophoresis tank, and then add the processed protein sample solution to the U-shaped groove formed on the plate rubber at 75 volts. Electrophoresis. After the electrophoresis, the protein was transferred, the colloid was taken out, and the colloid was spread on a soaked Whatman 3M filter paper. At this time, PVDF membrane (Polyvinylidene Fluoride Membrane) was previously covered with methanol. Colloid Above, cover a piece of soaked 3M filter paper in turn, and then use a glass rod to light the bubble in between, then load the Transfer Holder, and then place it in the Electrotransfer Tank (with transfer buffer) Printing buffer)) At 4 ° C, 100 volts voltage transfer, after 1 hour of electrotransfer, take out PVDF membrane (polytetrafluoroethylene film) immersed in 5% (w / v) skim milk (Blocking buffer) (PBS-non-fat milk powder, PBS-skimmed milk powder) was shaken at room temperature for one hour. The PVDF membrane (polytetrafluoroethylene membrane) was allowed to react with the primary antibody in a 4 degree refrigerator for 12 hours, and then washed twice with a Washing buffer, each time for 10 minutes, and finally washed again and then discarded. The Hordesadish peroxidase conjugated secondary antibody was further reacted for 2 hours to clean the PVDF membrane (polytetrafluoroethylene membrane) in the same manner. Finally, PVDF membrane (polytetrafluoroethylene membrane) was immersed in 4 ml of a substrate buffer for color reaction.

5. Cell survival analysis

The cells were cultured in a 24-well dish (24-well culture plate). After the cells were treated with the drug, the culture solution was removed and washed with PBS buffer (PBS solution) (3 times), and the culture medium containing 0.5 mg/ml MTT was exchanged. After about 3 to 4 hours, the culture solution was removed and washed with PBS buffer (PBS solution), and purple formazan (triphenylmethyl) crystal was dissolved by adding 1 ml of isopropanol (isopropanol), and the absorbance of OD570nm was measured after 5 minutes. .

6.DAPI (4,6–diamidino-2-phenylindole) cell fluorescence staining

After the cells were treated with drugs, the culture solution was removed and washed with PBS buffer (PBS solution) (3 times), and the cells were fixed with 4% paraformaldehyde (paraformaldehyde) for 30 minutes at room temperature, and then washed with PBS buffer (PBS solution). Paraformaldehyde (paraformaldehyde) was removed three times, DAPI (4,6-diamidino-2-phenylindole) (1 μg/ml) was added for staining for 30 minutes and then washed three times with PBS solution using a fluorescence microscope (340/380 nm Excitation). Wavelength observation, 100X photo archive.

7. Apoptosis analysis

After the cells were treated with the drug, the culture solution was removed and washed with PBS buffer (PBS solution) (3 times), and the cells were fixed with 4% paraformaldehyde (paraformaldehyde) for 1 hour at room temperature, and then washed with PBS buffer three times to remove paraformaldehyde ( Paraformaldehyde); followed by addition of permeabilisation solution (permeate, 0.1% Triton X-100in 0.1% sodium citrate) for 2 minutes at 4 ° C and then washed three times with PBS. The reaction was treated with a TUNEL reaction mixture (TUNEL reaction mixture, label solution + enzyme solution) for 1 hour, and the cells were observed by a fluorescence microscope (450-500 nm excitation) at a wavelength of 100X.

8. Green tea EGCG enhanced fat stem cell ability experiment

In the stem cell proliferation experiment, the number of colonies produced by stem cells under different experimental conditions is dominant, and the more colonies, the better the growth of stem cells under this experimental condition. The stem cells were divided into 5 groups: stem cell group (group 1), stem cell high glucose injury group (group 2), EGCG (2.5 μM concentration) pretreatment stem cell plus high glucose injury group (group 3), EGCG (5 μM concentration) pretreated stem cells plus high glucose injury group (group 4) and EGCG (10 μM concentration) pretreated stem cells plus high glucose injury group (group 5). As can be seen from Fig. 3, the number of colonies of the five groups was 338±38, 100±26, 152±17, 178±22 and 226±31, respectively. Compared with the normal group, when the stem cells were cultured in the high glucose, the colony distribution of the stem cell growth was inhibited (group 1 > group 2, p < 0.001). In contrast, compared with the damage of high glucose, the colony distribution of stem cells was restored under different concentrations of EGCG (group 2<group 3, p<0.05; group 2<group 4, p<0.05; Group 2 <group 5, p < 0.01).

Next, test the ability of stem cells to move under different experimental conditions. The more cells, the stronger the ability of stem cells to move under this experimental condition. We divided the stem cells into 5 groups, the stem cell group (group 1), the stem cell high glucose injury group (group 2), and the EGCG (2.5 μM concentration) pretreatment stem cell plus high glucose injury group (group 3). EGCG (5 μM concentration) pretreated stem cells plus high glucose injury group (group 4) and EGCG (10 μM concentration) pretreated stem cells plus high glucose injury group (group 5). Figure 4 is a test of stem cell mobility. The number of stem cell movements in these 5 groups was 63 ± 10, 36 ± 7, 55 ± 5, 84 ± 6, and 144 ± 4, respectively. Compared with the normal group, under the damage of high sugar, The ability of stem cells to move decreased (group 1 > group 2, p < 0.05). On the contrary, compared with the damage of high glucose, the migration ability of stem cells was restored under different concentrations of EGCG (group 2<group 3, p<0.05; group 2<group 4, p<0.001; group) Do not 2 <group 5, p < 0.001). Figure 5 shows the analysis of protein expression levels of stem cells under different experimental conditions. When analyzing the expression of protein, it was found that the expression level of stem cell mobile protein CXCR4 was decreased in high glucose injury compared with the normal group. On the contrary, compared with the damage of high sugar, the expression level of mobile protein CXCR4 and stem cells of stem cells recovered under different concentrations of EGCG. A similar situation can be observed in the expression level of the survival-related protein p-Akt. In the expression level of stem cell apoptosis protein cytochrome-C, compared with the normal group, the expression level of stem cell apoptosis protein cytochrome-C increased at high glucose damage. On the contrary, compared with the damage of high glucose, the expression level of apoptosis protein cytochrome-C in stem cells decreased under different concentrations of EGCG. Figure 6 shows the analysis of protein expression of stem cells under different experimental conditions. When analyzing the expression of protein, it was found that the expression of CXCR4 and p-Akt in stem cells increased by EGCG and disappeared due to the addition of siRNA CXCR4. Figure 7 shows the mobility test of stem cells, in order to test the ability of stem cells to move under different experimental conditions. This experiment divides stem cells into 6 groups, including stem cell group (group 1), stem cell plus high glucose injury group (group). 2), EGCG (10 μM) pretreatment stem cell plus high glucose injury group (group 3), CXCR4 siRNA (3 nM) in group 3 (group 4), CXCR4 siRNA (5 nM) in group 4 (Group 5) and siRNA (10 nM) added to CXCR4 in Group 5 (Group 6). The number of stem cell movements in these 6 groups were 288±25, 36±7, 159±17, 84±6, 41±8 and 40±2, respectively. The number of stem cell movements in group 2 was significantly less than that in group 1 (p<0.001), group 3 was more than group 2 (p<0.001), group 4 was less than group 3 (p<0.01), Group 5 was less than group 3 (p < 0.001) and group 6 was less than group 3 (p < 0.001).

The next experiment is to test whether the stem cells have a regenerative effect on H9c2 cardiomyocytes under the damage of high sugar. Figure 8 shows the expression of survival-related proteins in H9c2 cardiomyocytes under different experimental conditions. Compared with the normal group (column 1), under high glucose damage (column 2), survival-related proteins such as IGF1, PI3K, Akt And the expression level of p-Bad protein is decreased, and the table of these surviving proteins The up-regulation was observed in the regeneration of stem cells (column 3), and the addition of stem cells pretreated with EGCG (column 4) showed much more survival-related expression than untreated stem cells. Figure 9 shows the expression level of survivin p-Akt under different experimental conditions when H9c2 was co-cultured with stem cells. Addition of high glucose can reduce the expression of survivin in H9c2, while stem cells pretreated with stem cells or EGCG can increase the expression of survivin in H9c2 cardiomyocytes. After addition of CXCR4 siRNA, the regeneration of stem cells and EGCG pretreated stem cells can be achieved. The effect will be suppressed.

Example 2: Animal Experiment Design and Analysis

The 2-month-old Wistar strain male mice (purchased from Green Seasons Company) were divided into four groups, namely the normal group, the STZ (55 mg/kg)-induced diabetes group, the diabetes plus autologous adipose stem cell treatment group, and the diabetes plus green tea EGCG pretreatment. Autologous adipose stem cell treatment group, etc. The rats were housed in the animal room under the circulation of 12 hours during the day and 12 hours during the night. During the feeding period, the diet and drinking water were free to feed. Two rats were fed together with one animal cage, and the animals were changed every two days during the feeding period. When the blood glucose of the diabetic group rose to 200 mg/dl, it was diagnosed as having diabetes, and it was determined that the diabetic group was treated with autologous stem cell transplantation one month later. Autologous stem cell transfusion is the tail vein of each rat in a manner reinfusion Ke 1 × 10 ⁶ stem cells.

1. Animal serum and body weight analysis

The experimental rats were divided into 4 groups: normal group (sham), diabetic group (DM), stem cell treatment diabetes group (DM+ADSC) and EGCG pretreatment stem cell treatment group (DM+E-ADSC). Fig. 10(A) shows the blood glucose level (glucose in serum) in the blood of the sham group, the DM group, the DM+ADSC group, and the DM+E-ADSC group after the sacrifice of the animal after the experiment is completed. The values were 126±9 mg/dl, 611±35 mg/dl (p<0.01 vs. sham group), 493±37 mg/dl (p<0.01 vs. DM group) and 451±16 mg/dl (compared with DM group p). <0.01). In terms of body weight, as shown in Fig. 10(B), the body weights of sham group, DM group, DM+ADSC group and DM+E-ADSC group were 627±46g and 438±28g, respectively (with sham group). Ratio p<0.05), 473±6g and 477±13g.

2. Animal heart ultrasound analysis

Ultrasound analysis of animal heart was entrusted to the cardiologist of China Medical University according to the standard operating procedures in the hospital. Figures 11 to 12 show the cardiac ultrasound analysis of experimental rats. The purpose is to analyze the cardiac function of rats in different groups. . Figure 11 shows the echocardiographic analysis of the heart. The red arrow (black arrow) indicates the ability of the heart to contract. The longer the black arrow, the worse the contraction ability of the heart. Compared with the sham group, the black arrow of the DM group is longer, indicating that the DM group has a mouse. The contraction ability of the heart was worse than that of the sham group. The black arrows of the DM+ADSC and DM+E-ADSC groups in the treatment group were shorter than those in the DM group, which represented the cardiac contractility of the two groups of DM+ADSC and DM+E-ADSC. The DM group is good. Figure 12 (A) shows the blood ejection rate (EF%) of the heart. The higher the injection rate, the better the cardiac function. The blood ejection rates of the four groups are sham group, DM group, DM+ADSC group and DM+E-. The ADSC group was 75±4%, 52±5% (p<0.05 vs. sham group), 60±3%, and 68±1% (p<0.05 vs. DM group). Figure 12 (B) is the heart compression rate (FS%), the higher the heart compression rate, the better the heart function, the four groups of heart compression rate sham group, DM group, DM+ADSC group and DM+E-ADSC group They were 41±3%, 24±3% (p<0.05 vs. sham group), 28±2%, and 34±1% (p<0.05 vs. DM group).

The study of the hypertrophy pathway of animal heart tissue, the change of left ventricle means the change of heart function. Before the sacrifice of the animal, we performed a cardiac ultrasound examination on the rat to observe the effect of diabetes on the left ventricle and the regeneration effect of stem cells on the left ventricle. Fig. 18 to Fig. 20 show the observation results of the left ventricle of each group of mice by ultrasonic waves. Figure 18A is an observation of the ventricular septal thickness at end diastole (IVSd). The values between the groups are sham = 1.36 ± 0.1 mm, DM = 0.98 ± 0.2 mm, and DM + ADSC = 1.22 ± 0.1 mm. And DM+E-ADSC=1.2±0.1mm. Figure 18B is an observation of the ventricular septal thickness at end systole (IVSs). The values between the groups are sham = 2.68 ± 0.2 mm and DM = 1.49 ± 0.1 mm (p < 0.01 compared with the sham group). DM+ADSC=2.14±0.1 mm (p<0.05 compared with DM group) and DM+E-ADSC=2.26±0.3 mm (p<0.05 compared with DM group). Figure 19 (A) is to observe the left ventricular posterior wall thickness at end diastole (LVPWd), the values between the groups are sham = 1.36 ± 0.4 mm, DM = 0.85 ± 0.1 mm, DM + ADSC = 1.11 ± 0.2 mm and DM + E-ADSC = 1.12 ± 0.3 mm. Figure 19 (B) is the left ventricular posterior wall thickness at end systole (LVPWs), the values between the groups are sham = 2.19 ± 0.2 mm, DM = 1.3 ± 0.1 mm (and sham) Group comparison p<0.05), DM+ADSC=1.5±0.3 mm, and DM+E-ADSC=2.18±0.1 mm (p<0.001 compared with DM group). Figure 20 shows the hypertrophy-associated protein analysis of myocardial tissue. Compared with the sham group, the protein expression levels of hypertrophic related proteins such as p-GATA4, ANP and BNP were significantly increased in the DM group; in the treatment group DM+ADSC and DM+E The expression of hypertrophy-related protein in ADSC was significantly lower than that in DM group, especially in DM+E-ADSC group. Conversely, the expression level of p-NFATc3, a non-hypertrophic factor, is inversely proportional to the expression of hypertrophic factors.

3. Heart tissue sectioning, staining and analysis

Cardiac tissue sectioning, staining and analysis were performed by the Department of Pathology of Changhua Christian Hospital. The experimental rats were sacrificed after the end of the animal experiment, and the heart tissue was taken for sectioning and staining. The purpose was to observe the arrangement of myocardial cells and myocardial The size of the tissue gap. If the heart is damaged, the arrangement of cardiomyocytes will be disordered and the myocardial tissue gap will become larger. Figure 13 is a staining analysis of HE slices in animal heart tissue. Compared with the sham group, the heart tissue sections of the DM group showed that the arrangement of myocardial cells (blue dots) was disordered and the myocardial tissue gap became larger (white space); Compared with the DM group, the heart tissue sections of the treatment group (DM+ADSC and DM+E-ADSC) showed that the arrangement of the cardiomyocytes (blue dots) was neat and the myocardial tissue gap became smaller (white space).

Example 3: Animal cardiomyocyte cell culture and analysis

Embryonic rat myocardial transformed cell line H9c2cells (from ATCC CRL-1446) and adipose stem cells cultured in 10% fetal bovine serum (fetal bovine serum, FBS, Hyclone), 1% Antibiotic-Antimycotic (antibacterial-antimycotic, Gibco) Dulbeco's Modified Eagle Medium (DMEM, Sigma), incubator set 5% CO2, 37 ° C constant temperature environment, change the accompanying liquid 2 to 3 times a week. Cardiomyocytes were cultured at different time points or at different drug concentrations using culture-free medium (serum-free medium).

1. Analysis of animal cardiomyocyte survival protein

After the end of the animal experiment, the hearts of the rats in each group were freed and homogenized, and the expression levels of survival-related proteins in the heart tissue of each group were analyzed by Western blotting method. As can be seen from Fig. 14, compared with the sham group, the expression of protein related to survival in the DM group was significantly decreased; while in the treatment group, the expression of survivin protein in the DM+ADSC and DM+E-ADSC groups was significantly higher than that in the DM group. High; further observations showed that the expression levels of the three survival-related proteins IGF1R, p-PI3K and p-Akt in the DM+E-ADSC group were much higher than those in the DM+ADSC group.

2. Analysis of animal cardiomyocyte apoptosis protein

After the end of the animal experiment, the hearts of the rats in each group were freed and homogenized, and the expression levels of apoptosis-related proteins in the heart tissues of each group were analyzed by Western blotting method. As can be seen from Fig. 15, compared with the sham group, the expression of apoptosis-related proteins in the DM group was significantly increased; while in the treatment group, the expression levels of apoptotic proteins in the DM+ADSC and DM+E-ADSC groups were significantly higher than those in the DM group. The group was low; further observation revealed that the expression level of apoptosis-related proteins in the DM+E-ADSC group was lower than that of the DM+ADSC group. The apoptosis of cardiomyocytes was observed by TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining. The staining was performed by two dyes TUNEL and DAPI, and DAPI was the nucleus of cardiomyocytes. ) is used to confirm the number of cardiomyocytes; while TUNEL is an apoptotic cardiomyocyte (showing green), that is, apoptotic cardiomyocytes are detected by green fluorescence. The apoptotic signal (green) in the four groups of sham, DM, DM+ADSC and DM+E-ADSC were 2±1%, 14±4%, 6±1% and 5±2%, respectively (Figure 16 ). Compared with the sham group, the green fluorescence highlights of the DM group were significantly more (sham<DM, p<0.01), while the treatment group DM+ADSC (DM>DM+ADSC, p<0.05) and DM+E-ADSC (DM) The green fluorescence high point of >DM+E-ADSC, p<0.05) was significantly less than that of the DM group.

3. Analysis of Sirt1 related proteins in animal cardiomyocytes

After the end of the animal experiment, the hearts of the rats in each group were freed and homogenized, and the expression levels of Sirt1-related proteins in the heart tissues of each group were analyzed by western blotting method. As can be seen from Figure 17, compared with the sham group, the expression of Sirt1-related protein in the DM group was significantly decreased. In the treatment group, the expression levels of Sirt1 protein in the DM+ADSC and DM+E-ADSC groups were significantly higher than those in the DM group. High; further observation revealed that the expression level of Sirt1-related protein in the DM+E-ADSC group was higher than that of the DM+ADSC group.

4. Discussion on the path of fibrosis in animal heart tissue

At the end of the animal experiment, the rats were sacrificed, and the heart tissue was taken and stained with Masson Trichrome (triple staining) to observe the accumulation of collagen in the blue part of the heart tissue. If the area of the blue part is larger, the more collagen is accumulated, the more severe the condition of cardiac fibrosis. Figure 21 shows the results of Masson Trichrome staining of heart tissue in each group. Compared with sham, the blue collagen accumulation in the DM group was significantly increased, compared with the DM group, the treatment group DM+ADSC and DM+E. - The blue collagen accumulation portion of ADSC is significantly less. Figure 22 shows the expression of fibrosis-associated protein after homogenization of cardiac tissue. Figure 22 shows that compared with sham, the expression of fibrotic protein in DM group increased significantly; compared with DM group, treatment group DM +ADSC and DM+E-ADSC two groups of fibrosis-related protein expression decreased significantly

According to the above relevant research data, diabetes can cause damage to heart tissue of rats, and adipose stem cells have the ability to restore the regeneration of diabetic heart tissue. If fat stem cells are treated with green tea EGCG, the fat of green tea EGCG is pretreated. Stem cells have a significant increase in the ability to regenerate heart damage caused by diabetes. It can be seen that the pretreatment of adipose-derived stem cells with green tea EGCG can enhance the regeneration ability of stem cells. It is known from cell experiments that green tea EGCG can increase the expression of CXCR4 protein on the adipose-derived stem cell membrane, while the expression of CXCR4 increases, the stem cell Proliferative capacity, viability, anti-apoptosis ability and mobility have all increased significantly. Further data were obtained in animal experiments to confirm the regeneration of stem cells treated with green tea EGCG. The ability of myocytes is better than that of cardiomyocytes regenerated from stem cells that have not been treated with green tea EGCG. Figure 23 illustrates how green tea EGCG increases the ability of adipose stem cells to regenerate cardiomyocytes by increasing the amount of CXCR4 expressed.

The present invention demonstrates that green tea EGCG can increase the expression of CXCR4 to enhance the regeneration of diabetic myocardial function damage by adipose stem cells. In the future, when stem cells are used for clinical purposes, there is often a problem of stem cell reinfusion dose, if green tea EGCG can be used. When the stem cells are treated, the therapeutic ability of the stem cells can be improved under the dose limitation of the stem cell reinfusion, and the treatment of the stem cells is more remarkable.

Claims (15)

1. A pharmaceutical composition for treating heart disease, characterized in that the pharmaceutical composition comprises adipose stem cells.
2. The pharmaceutical composition according to claim 1, wherein the heart disease is heart disease caused by diabetes or hyperglycemia.
3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises an ester catechin.
4. The pharmaceutical composition according to claim 1, wherein said adipose stem cells are pretreated with ester catechin for 2 hours.
5. The pharmaceutical composition according to claim 4, wherein the pretreated adipose stem cells have a concentration of ester catechin of 5-15 μM.
6. The pharmaceutical composition according to claim 4, wherein the pretreated fat stem cells have a concentration of the ester catechin of less than 20 μM.
7. A method of preparing a pharmaceutical composition for treating heart disease, characterized in that the pharmaceutical composition comprises 1 x 10 ⁵ adipose stem cells treated with ester catechin.
8. The method of claim 7 wherein said heart disease is caused by diabetes or hyperglycemia.
9. The method according to claim 7, wherein 1 × 10 ⁵ adipose stem cells treated with ester catechin are administered to said individual by intravenous injection.
10. The method according to claim 9, wherein said adipose stem cells are pretreated with ester catechin for 2 hours and then administered to said individual by intravenous injection.
11. The method according to claim 10, wherein said pretreated adipose stem cells have a concentration of ester catechin of 5-15 μM.
12. The method of claim 10 wherein said pretreated adipose stem cells have a concentration of ester catechin of less than 20 [mu]M.
13. A method for enhancing tolerance and mobility of stem cells to sugars, characterized in that the method comprises adding an ester catechin to a medium of stem cells.
14. The method of claim 13 wherein said ester catechin has a concentration of 5-15 μM.
15. The method according to claim 13, wherein the method further comprises removing the stem cells in a medium containing the stem cells of the ester catechin for 2 hours.

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