Impact Of Tbx3 On Differentiation Of Murine Cardiac Stem Cells

Objective:To preliminarily explore possible role of Tbx3 in differentiation of murine CSCs towards pacemaker-liked cells by upregulating Tbx3 expression with overexpression lentivirus.Methods:1.Primary CSCs isolation,cultivation,and passage:got the heart tissue of Kunming mouse.Explant culture method was applied to acquire CSCs.When confluency of murine CSCs inculture dish reached 70%,collecting them for further experiment;2.Construction of Tbx3 gene over-expression lentiviral vector:DNA fragment of human Tbx3 which was purchased from gene company was amplified by PCR,digested with Xba I,BamH I and cloned into the lentiviral vector pLenti-CMV-3FLAG-EGFP.The positive clones were selected to be submitted to DNA sequencing.The identification DNA sequencing confirmed that Tbx3 lentiviral vector was successfully constructed;3.Production of Tbx3 over-expression lentivirus package and titer detection:transfected 293T by aforementioned vectors,harvested virus-containing supernatants 48-72 hours after transfection,improved the purity and then titered the lentiviral stock by RT-PCR;4.Detection of HCN2,HCN4 expression in murine CSCs at mRNA level(with RT-PCR)3 days after infection and at protein level(with Western-blot)7 days after infection with Tbx3 over-expression lentivirus.Results:1.Primary cultivation of murine CSCs with explant culture method:4h after being planted,myocardial tissue adherent appeared.5-7d later,murine CSCs that were round,high refraction,dispersion and anucleate creep,which adhered to the fibroblasts.14d later,confluency of murine CSCs in dishes reached approximately 70%.Minority of them appeared to differentiate;2.Tbx3 over-expression lentiviruses were successfully constructed,the result of titer was 2.09×108 TU/ml;3.expressing of Tbx3 over-expression lentivirus in CSCs:Western-blot showed a positive band at 84KD 72h after infecting at MOI70,suggesting that Tbx3 over-expression lentivirus expressed correctly in CSCs;4.RT-PCR showed a significant increase of HCN4 expression in Tbx3 over-expression lentivirus infected murine CSCs,compared with control group(P<0.05),while HCN2 expression was not increased significantly by Tbx3 over-expression lentivirus infection(P>0.05);5.Western-blot showed no detectable HCN2,HCN4 expression.Conclusion:1.It is reliable and stable to acquire murine CSCs with explant culture method.2.MOI70 is the optimum level for CSCs infection.3.Overexpressing of Tbx3 in murine CSCs can up-regulate the expression of HCN4.4.Tbx3 is not able to promote CSCs differentiate into pacemaker-liked cells.