The Effect Of Bmp13 On C3H10T1/2 Cells Differentiate Into Cardiomyocyte-Like Cells And Cardiac Function In Myocardial Infarction Rats

Part one: To obtain high titer of AdBMP13 and AdGFP and observe the growth and differentiation of Transfected C3H10T1 / 2 cell.Objective: To obtain high titer of AdBMP13 and AdGFP by Cultivate and transfect the 293 cells, infect the C3H10T1 / 2 cells with AdBMP13 to overexpress the BMP13, and the growth and differentiation of target cells were observed.Methods: 293 cells culture and the low titer AdBMP13 repeated AdGFP adenovirus amplification in these cells, high titer adenovirus were collected. When C3H10T1 / 2 cells passaged to 60% to 70%, add the high-titer adenovirus to infect the C3H10T1 / 2 cells and the medium was changed after 6 hours, fluorescence microscope expression of green fluorescence and infection efficiency by flow cytometry 24 hours after the infection.The cardiac structure and function of protein a-MHC and cTNT expression of the cells were observed on week 1, week 2, week 3, week4 by immunofluorescence. Results: 1.The virus stock solution was amplified in 293 cells after repeat and obtained a high titer AdBMP13 and AdGFP at 5.0 ~ 6.0×1010 /μL.2. C3H10T1 / 2 cells were infected by high-titer AdBMP13 and AdGFP and the expression efficiency of fluorescent flow cytometry were 89.42% and 88.41% after 24 hours.3. The cells shape was from polygonal, spindle to the long strip over time by inverted microscope morphology.4. There were significantly different between GFP group and BMP13 group on cardiac structure and function related to a-MHC protein expression and cTNT by immunofluorescence.Conclusion: We could obtain the high titer adenovirus by 293 cells; there was high infection rate in cell transfection; the transfected C3H10T1 / 2 cells were in good conditions and could differentiate into cardiomyocyte-like cells. Part two: C3H10T1 / 2 cells transfected with BMP13 to improve cardiac function in myocardial infarction rats.Objective To investigate the bone morphogenetic protein 13 (BMP13/GDF6) could promote C3H10T1/2 cell differentiation into cardiomyocytes and improve the cardiac function of the rats after myocardial infarct (MI) in microenvironment.Methods MI and sham operation were established Sprague Dawley (SD) male rats. Totally 56 rats were divided into 4 groups: sham operation group(8 rats), MI group(16 rats), MI+GFP group(16 rats), MI+BMP13 group(16 rats). Left ventricular(LV)function were assessed by echocardiography on day baseline and 4 weeks after operation.After measuring body weight once a week, 4 weeks after completion of cardiac function test, rats were sacrificed to take the heart. Masson's staining compared the differences in infarct size of heart, immunofluorescence detection cardiac-specific structural protein a-MHC and cTNT of cells were implanted in each group.Results We observed the better cardiac function in MI + BMP13 group and MI + GFP group compare with MI group were reduced(P <0.05),the former was better than the latter. The implanted cells in group MI + GFP and MI + BMP13 had cardiac cell-specific structural protein a-MHC and cTNT expression by immunofluorescence. Conclusion C3H10T1/2 cells can grow and differentiation into cardiomyocyte-like cells in micro-environment of myocardial infarction. BMP13 can induce differentiation of C3H10T1/2 cells into cardiomyocyte-like cells and improve cardiac function in myocardial infarction rats.