| At present,the morbidity and mortality of cardiovascular disease（CVD）continue to increase,which seriously endangers human life and health.Acute myocardial infarction（AMI）is the most common and serious cardiovascular disease.AMI results in necrosis of myocardial cells that affects cardiac function.Although clinical treatment methods can improve the prognosis of myocardial infarction such as coronary artery bypass grafting,percutaneous coronary intervention and drugs,they cannot repair and regenerate damaged myocardium.Stem cells have characteristics such as multidirectional differentiation potential and self-renewal ability,which provide a new idea for regeneration and repair of infarcted myocardium and reversing myocardial damage.As one of the basic biological elements of human tissue and organ regeneration,stem cells have received increasing attention as therapeutic methods for repairing damaged tissues and organs.Among them,bone marrow mesenchymal stem cells（BMSCs）have become a research hotspot due to their characteristics of easy cultivation,strong plasticity and low immunogenicity.BMSCs can differentiate into cardiomyocytes or cardiomyocyte-like cells under appropriate induction conditions,obtaining myocardial-like phenotypes.However,in clinical practice,there is a problem of poor differentiation after transplantation of BMSCs,so selecting suitable inducers becomes extremely important.In recent years,resveratrol has attracted much attention due to its protective effects on the cardiovascular system.Resveratrol is a polyphenolic compound produced by plants with a variety of biological activities,including inhibition of myocardial injury,anti-myocardial fibrosis and anti-inflammatory effects.However,excessive resveratrol concentration can also inhibit the cell activity of BMSCs.It has been found that appropriate concentrations of resveratrol can promote embryonic stem cells（ESCs）and induced pluripotent stem cells（i PSCs）to differentiate into cardiomyocytes,so as to improve the differentiation efficiency of cardiomyocytes.This experiment used different concentrations of resveratrol to induce BMSCs in vitro,to explore whether resveratrol can promote the differentiation of BMSCs into cardiomyocyte-like cells from various aspects and optimal inducing concentration of resveratrol.At the same time,to explore the possible mechanism of resveratrol promoting the differentiation of BMSCs into cardiomyocyte-like cells,This study provides experimental basis for improving the induced differentiation rate of BMSCs and theoretical support for personalized treatment of patients.To investigate the feasibility and possible mechanism of resveratrol in inducing differentiation of BMSCs into cardiomyocyte-like cells,this experiment used whole bone marrow adherent method to extract the rat bone marrow cells and used flow cytometry to identify the surface antigens of the third generation of BMSCs;Cell Counting Kit-8（CCK-8）was used to detect the effects of 5,10,20,50,100,200μmol·L-1resveratrol on the cells viability of BMSCs;Real-time quantitative PCR（RT-q PCR）was used to detect the expression of GATA-4,Nkx2.5 m RNA in BMSCs induced by different concentrations of resveratrol at 1 week,2 weeks and 4 weeks;Immunocytochemical staining was used to detect the expression of connexin43（Cx43）,cardiac troponin T（c Tn T）and cardiac troponin I（c Tn I）in each group after 4 weeks of induction;Western Blot was used to detect the expression of Desmin and c Tn T in each group after 4 weeks of induction;Cells were divided into control group,10μmol·L-1 resveratrol group and10μmol·L-1 resveratrol+Lithium chloride（Li Cl）group.Western Blot was used to detect the changes of p-β-catenin/β-catenin in Wnt/β-catenin signaling pathway of each group.The morphological changes of BMSCs during the culture process were observed under an inverted phase contrast microscope,the cell morphology changed from the initial round to triangular,short spindle,irregular and long spindle.After induction with 10μmol·L-1resveratrol,spindle cells decreased,triangular and irregular cells were observed.Subsequently,cell junctions appeared between the cells,some cells were long columns or polygons,the cells were arranged closely and directional.There was no significant difference in the morphology of BMSCs induced by different concentrations of resveratrol.Flow cytometry showed that the positive expression rates of CD29,CD90 and CD45 were 91%,95%and 2.4%,respectively.CCK-8results showed that 50,100,200μmol·L-1 resveratrol inhibited the cell viability of BMSCs,while 5,10,20μmol·L-1 resveratrol had no effect on the cell viability of BMSCs and the difference was statistically significant（P<0.05）.RT-q PCR results showed that the relative gene expression of GATA-4 in the 5,20μmol·L-1 resveratrol group was the strongest at 4 weeks,while the expression of GATA-4 in the 10μmol·L-1 resveratrol group reached a peak at 2 weeks.The time point was 2 weeks after induction,the 10μmol·L-1 resveratrol group was significantly higher than the other groups and the difference was statistically significant（P<0.05）.The relative gene expression of Nkx2.5 in the 5μmol·L-1 resveratrol group was weaker at different time points,while the expression of Nkx2.5 in the 10μmol·L-1resveratrol group increased with time.The time point was 4 weeks after induction,the 10μmol·L-1resveratrol group was significantly higher than the other groups and the difference was statistically significant（P<0.05）.Immunocytochemical staining results showed that Cx43,c Tn T and c Tn I were positive in all the induced groups,the 10μmol·L-1 resveratrol group had the highest positive expression,the difference was statistically significant（P<0.05）.Western Blot results showed that the expression of Desmin protein in 5,20μmol·L-1 resveratrol group was not significantly different from in the control group,while the expression of Desmin protein in 10μmol·L-1resveratrol group was significantly higher than the control group（P<0.05）.The expression of c Tn T protein in the 5,10,20μmol·L-1 resveratrol group was higher than the control group and the protein expression of 10μmol·L-1resveratrol group was the highest.the difference was statistically significant（P<0.05）.Western Blot was used to detect the change of p-β-catenin/β-catenin,the results showed that the p-β-catenin/β-catenin in the control group and 10μmol·L-1 resveratrol+Li Cl group was significantly lower than that in the 10μmol·L-1 resveratrol group（P<0.05）.The above experimental results indicate that resveratrol can induce BMSCs to differentiate into cardiomyocyte-like cells under certain conditions,10μmol·L-1 may be the optimal inducing concentration.Resveratrol may promote the differentiation of BMSCs into cardiomyocyte-like cells by inhibiting the expression ofβ-catenin protein in Wnt/β-catenin signaling pathway. |
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