Effects Of Wnt-11 Combined With 1,25-Vitamin-D3 On Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Cardiomyocytes

The occurrence of cardiovascular disease?CVD?,such as acute myocardial infarction?AMI?,is a serious threat to the health of the middle-aged and the elderly.In recent years,there has been a tendency to be young.Complications associated with AMI greatly reduce people's quality of life.Although some studies have shown that human cardiomyocytes have the ability to divide and proliferate to some extent,the renewal rate of only 1%per year after the occurrence of AMI is difficult to make up for the number of damaged cardiomyocytes and can not effectively improve the cardiac function.At present,the main clinical treatment methods for myocardial infarction,such as drug therapy,thrombolytic therapy,interventional therapy and surgical treatment,are all aimed at saving myocardial cells in non-infarct areas.However,there is a lack of fundamental treatment for myocardial reconstruction and regeneration in patients with infarction.Based on this status quo,some scholars have suggested that exogenous cells can be transplanted into the injured myocardium to restore and improve the function of myocardium by a sufficient amount of cells with the contractive function.At present,stem cell research has become the most attractive field of natural science.There are a variety of cells,such as embryonic cancer cell line?P19?,embryonic stem cells,adipose-derived stem cells,bone marrow stromal cells,have been used to induce differentiation and then transplant to treat myocardial infarction.Autologous bone marrow mesenchymal stem cells?BMSCs?can proliferate in suitable culture environment in vitro,showing strong differentiation plasticity and producing corresponding cell markers,which have become an ideal seed cell in the field of tissue engineering.Current studies have confirmed that both Wnt-11 and 1,25-vitamin-D₃play the important role in cardiac development and cardiomyocyte differentiation,and both of them play an important role by inhibiting the classical Wnt signaling pathway.1,25-vitamin-D₃ could also promote the expression of Wnt-11.Therefore,BMSCs were induced to differentiate into cardiomyocytes by the combination of them and alone in this study.The effects of Wnt-11 and 1,25-vitamin-D₃ on their proliferation and differentiation were investigated from two aspects of morphology and molecular biology.The best way to induce differentiation was selected to improve the differentiation rate of cardiomyocytes in order to provide a theoretical basis for cell transplantation treatment of ischemic heart disease.The BMSCs of SD rats were isolated and cultured by whole bone marrow adherent method and density gradient centrifugation.The second generation of BMSCs was induced by Wnt-11 combined with1,25-vitamin-D₃ and both alone.Under the inverted phase contrast microscope,most of the primary cells showed a short spindle shape after 72hours of culture.After 1 week of culture,the cells showed diversified morphology.The adjacent cells of BMSCs,induced after 4 weeks,were closely connected with each other,and the arrangement had obvious directivity.BMSCs were identified by flow cytometry.The positive expression rates of CD29,CD45,CD90 were 97.4%,3.3%and 91.4%,respectively.Immunocytochemistry,immunofluorescence cytochemistry and Western blot were used to detect the expressions of tropomyosin?TPM?,connexin 43?Cx43?and cardiac troponin T?cTnT?in BMSCs at 4 weeks after induction.The results showed that the positive expressions of TPM,Cx43 and cTnT were found in BMSCs of each group,and the combination induction group was the highest,while the uninduced group cells showed a weak positive or negative expression The difference was statistically significant?P<0.05?.Transmission electron microscopy?TEM?was used to observe the ultrastructure of differentiated cells similar to that of cardiomyocytes.TEM showed that many organelles,such as myofilaments and mitochondria,ribosomes and rough endoplasmic reticulum,could be seen in the cytoplasm of cultured cells at 4 weeks after induction.The early transcription factor GATA-4 and Nkx2.5 were detected by real-time quantitative polymerase chain reaction?RT-qPCR?at 1,2 and 4weeks after induction.The results of RT-qPCR showed that the induced cells could express GATA-4 and Nkx2.5 at the 1st week,and then the expression of them decreased at the 2nd week,but then increased at the 4th week.In terms of gene expression,the combined induction group was significantly better than the single induction group?P<0.05?,and the gene expression of uninduced group was 1.Through this experiment,it can be explained that:1.BMSCs isolated from SD rats by whole bone marrow adherent method and density gradient centrifugation could be induced to differentiate as seed cells in tissue engineering field.2.Rat BMSCs cultured in vitro could differentiate into cardiomyocytes after being induced by Wnt-11,1,25-Vitamin-D₃ and the combination of them.3.In the process of inducing the differentiation of rat BMSCs into cardiomyocytes,the effect of Wnt-11 combined with 1,25-Vitamin-D₃ was significantly better than that of Wnt-11 or 1,25-Vitamin-D₃ alone.