Research On The Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Myocardial Like Cells Induced By AngⅡ Combined With FGF-2

As people’s living standards continue to improve,unhealthy lifestyle and diet have led to a steady increase in cardiovascular disease risk factors.Ischemic heart disease has become one of the leading causes of death worldwide and a major public health problem worldwide.Although conventional treatment can improve patient survival in clinical practice,however,considering the complexity of the disease,it is still necessary to explore other effective treatment methods.BMSCs are a widely distributed class of cells that retain the same capacity for self-renewal and multidirectional differentiation as other stem cells as one of the stem cell types.In addition to being free from social and ethical constraints,it has the advantages of paracrine secretion and promoting angiogenesis,and has leapt to become one of the more widely studied stem cell types.In vitro studies have shown that BMSCs can differentiate into cardiomyocyte,providing an alternative cell source for cardiovascular regeneration.There are multiple inducers in the differentiation of BMSCs to cardiomyocyte-like cells.Among them,cytokines are biologic factors with natural affinity and are the more widely studied inducers.Angiotensin Ⅱ(AngⅡ),as a hormone present in the body,plays a role as a growth factor and can promote the proliferation and differentiation of BMSCs.Fibroblast Growth Factor-2(FGF-2)is expressed in all stages of heart development.It can not only promote angiogenesis,but also protect the heart.FGF-2 promotes the proliferation of bone marrow mesenchymal stem cells by activating the ERK1/2 signaling pathway,and Ang Ⅱ can also induce the differentiation of human bone marrow mesenchymal stem cells into cardiomyocytes through this signaling pathway.However,there is currently no report on whether Ang Ⅱ combined with FGF-2 can improve the efficiency of BMSCs differentiating into myocardial like cells and whether the ERK1/2 pathway is involved in this process.There is currently no report on whether Ang Ⅱ combined with FGF-2 can improve the efficiency of BMSCs differentiating into myocardial like cells and whether the ERK1/2 pathway is involved in this process.Therefore,this experiment explores whether Ang Ⅱ combined with FGF-2 can more efficiently induce BMSCs to differentiate into myocardial like cells from morphological and molecular biology perspectives,and preliminarily explores the induction mechanisms of the two cytokines.This experiment first used the whole bone marrow adhesion method to cultivate BMSCs,and used flow cytometry to identify the surface antigens of the third generation BMSCs.Take well growing third generation cells and induce BMSCs with Ang Ⅱ and FGF-2 alone or in combination,while setting up a blank control group,and immunofluorescence was used to detect the expression of c Tn I in each group after 4 weeks of cultivation.The expression level of Nkx2.5 in different groups was detected by RT-q PCR technology at one week,two week,and fourth week of cultivation.Finally,Western blot was used to detect the expression levels of Cx43,c Tn T and Desmin in each group after 4 weeks of culture.The aim is to elucidate the feasibility of Ang Ⅱ combined with FGF-2 inducing BMSCs to differentiate into myocardial like cells.To further verify whether the ERK1/2 pathway is involved in this differentiation process,using Western blot method detect the expression levels of RAS,P-MEK,and P-ERK1/2 in the blank group and Ang Ⅱ combined with FGF-2 induction group for 4weeks.The cell growth and morphological changes were observed at different periods under the inverted microscope.It was observed that the cells were initially extracted in suspension,then gradually adhered to the wall at 24 hours,then irregularly shaped such as short shuttle at 72 hours and long shuttle at 7 days,and the cells were tightly arranged and had obvious directionality after 4 weeks of induction.The results of flow cytometry showed that the positive expression rates of CD45 was 2.5%,CD29 was91.1% and CD90 was 95.1%,that is indicating that the cells extracted in this experiment were BMSCs.Immunofluorescence and Western blot detection showed that compared with the blank control group,myocardial specific biomarkers c Tn I,c Tn T,Cx43,and Desmin were expressed in all induction groups,and the Ang Ⅱ and FGF-2 combination group had the highest expression level,and the difference was statistically significant(P<0.05).Indicate that the combination of Ang Ⅱ and FGF-2 can promote more efficient differentiation of BMSCs into myocardial like cells.RT-q PCR results showed that after one week of induction,each group began to express Nkx2.5,and the expression level gradually increased in the second week,with the highest expression level in the fourth week,compared with other groups,the Ang Ⅱ combined with FGF-2 group had the highest expression level,and the difference was statistically significant(P<0.05).It suggests that BMSCs enter the myocardial differentiation.Western blot detected a higher positive expression rate of RAS,p-MEK,and p-ERK1/2 in the Ang Ⅱ and FGF-2 combined induction group compared to the blank control group,and the difference was statistically significant(P<0.05),The combination of Ang Ⅱ and FGF-2 may promote the differentiation of BMSCs into myocardial like cells,which may be related to the ERK1/2 pathway.Experiments showed that the rat bone marrow MSCs cultured in vitro with Ang Ⅱ,FGF-2,and the combination of both could induce differentiation of BMSCs into myocardial like cells,and the combined induction effect was better than that of single induction;The ERK1/2pathway may be involved in the process of Ang Ⅱ combined with FGF-2induced directional differentiation of BMSCs into cardiomyocytes.