Effect Of Bone Morphogenetic Protein9 And Wnt3A Induce C3H10T1/2 Cells Differentiation To Cardiomyocyte-Like Cells In Vitro

PART ONE:C3H10T1/2 CELLS WERE INFECTED BY RECOMBINANT ADENOVIRUS GFP, WNT3AAND BMP9Objective:To detect the cell morphology and the stability of transfected efficiency of C3H10T1/2 cells after infected by adenovirus GFP, Wnt3a and BMP9.Methods:HEK293 cells were cultured from 70 to 80 percent and used to amplified high-titer recombinant adenovirus GFP, Wnt3a and BMP9. The cell suspension could be collected after observing the variety morphology of HEK293 cells untill cultured 48-72h. The cell morphology and the expression of green fluorescent protein were observed by inverted and fluorescence microscope and flow cytometry in C3H10T1/2 cells induced with GFP, Wnt3a and BMP9 48 hours after transfection.Results:1. High-titer recombinant adenovirus GFP, Wnt3a and BMP9 were obtained by HEK293 cells after repeatedly amplified.2. The morphology of C3H10T1/2 cells after being transfected by BMP9 and Wnt3a and BMP9 co-induction observed by inverted microscope after transfected 1 week was gradually from polygon to long spindle side, with tight connection. While cells induced by Wnt3a alone were arranged in disorder with long spindle growth.3. The transfection efficiency of cells after transfected 48 hours by AdGFP, AdWnt3a and AdBMP9 was about 60 percent detected by flow cytometry.Conclusion:Recombinant adenovirus GFP, Wnt3a and BMP9 can amplified by HEK293 cells, which can transfect C3H10T1/2 cells with high stable efficiency.PART TWO:THE DIFFERENTIATION OF WNT3A AND BONE MORPHOGENETIC PROTEIN9 INDUCE C3H10T1/2 CELLS INTO CARDIOMYOCYTE-LIKE CELLS IN VITROObjective:To investigate the effect of Wnt3a alone and combined with BMP9 on cardiomyocyte-like cells differentiation of C3H10T1/2 cells.Methods:C3H10T1/2 cells were randomly divided into 5 groups: ①control group, ②GFP group, ③Wnt3a group,④BMP9 group, ⑤Wnt3a and BMP9 co-induction group.The expression of cardiac-specific proteins Cx43 and cTnT in varies groups were analyzed by Western blot, immunofluorescence. Quantificational real-time polymerase chain reaction (q-RT-PCR) was used to detect the expression of cardiac-specific genes GATA4 and MEF2C 3 weeks after transfection.Results:1.The green fluorescent protein can still be stable expressed by C3H10T1/2 cells after 3 weeks transfected by adenovirus.2. The expression of proteins Cx43, cTnT and genes GATA4, MEF2C were similar among Wnt3a group, GFP group and control group, but significantly increased when co-induced by Wnt3a and BMP93 weeks after transfection.3. The expression of Cx43, cTnT, GATA4 and MEF2C showed no significant difference between BMP9 group and Wnt3a and BMP9 co-induction group.Conclusions:The induction of Wnt3a alone can not promote the differentiation of C3H10T1/2 cells to cardiomyocyte-like cells. But Wnt3a and BMP9 co-induction group can promote the differentiation.