Research On Wnt5b Induced BMSCs Differentiation Into Cardiomyocyte-like Cells

Objective: With the high mortality rate, coronary heart disease(CHD) has currently become a major life-threatening disease. Conventional treatments can’t regenerate the functional cell efficiently. Stem cell therapy for myocardial infarction is very promising, but there are still many problems, such as low differentiation rate, low survival rate after transplantation. This study intends to regulate the expression of wnt5 b, and investigate the rate of BMSCs differentiation into cardiomyocyte-like cells, aiming at providing a more effective method for treatment of myocardial infarction.Method:Bone marrow mesenchymal stem cells were isolated from bone marrow of 4 weeks SD male rat. BMSCs was cultured by adherent method, identified CD29,CD44,CD34,CD45 with flow cytometry(FCM) at the passage 3. Choosing BMSC which grow well divided into four experimental groups, transfecting si RNA-NC, si RNA-wnt5 b, PEX1-NC, PEX1-wnt5 b. In vitro, in 3 days, 7 days, 14 days we detected cell as follows: detecting m RNA of cardiac-specific factors Nkx2.5, GATA-4, CX43, Myo D by q PCR; detecting the expression of specific CX43, Desmin expression by immunofluorescence; detecting the expression of Desmin, c Tn I by Western Blot. Established model of myocardial infarction, and injected cells after 48 h, we detected cardiac function echocardiography after 1day and 28 days.After 28 days, we remove the heart and do HE staining, Masson staining, immunofluorescence.Result: Elongated cells were observed, showing polar growth, cell surface antigens were identified by FAC, such as CD29, CD44 positive, CD 34, CD 45 negative. After 3 days,q PCR detection was applied to cardiac-specific factors, Nkx2.5, GATA-4 expression levels increased in group, while inhibited in PEX1-wnt5 b group. There are significant differences comparing with the corresponding negative control group. CX43, Myo D expression decreased in si RNA-wnt5 b, while increased in PEX1-wnt5 b, and there are no significant differences comparing with the corresponding negative control group. After 7 days and 14 days, Nkx2.5, GATA-4, CX43, Myo D expression levels increased in group, while inhibited in PEX1-wnt5 b group. There are significant differences comparing with the corresponding negative control group.In vitro expression of CX43, Desmin, c Tn T increased in si RNA-wnt5 b group, while decreased in PEX1-wnt5 b group by immunofluorescence and western blot. Establishing MI model, we injected BMSCs into the infarcted myocardium surface, si RNA-wnt5 b group rats’ EF% are better than PEX1-wnt5 b group; we analyzed the infarct size by Masson staining: after infarct, si RNA-wnt5 b group had the largest fiber architecture area, while PEX1-wnt5 b group had the smallest. We could see statistical difference compared with corresponding groups. The result of immunofluorescence showed that CX43 expression in si RNA-wnt5 b group increased, while decreased in PEX1-wnt5 b group.Conclusion:wnt5b have an important role to cardiomyocyte-like cells in BMSCs.Overexpression Wnt5 b will inhibit MSCs differentiation into cardiomyocyte-like cells,and silence wnt5 b will promot their differentiation; wnt5 b induces BMSCs into cardiomyocyte-like cells possibly by altering the expression of GSK-3β.