Transfection Of Adipose Stem Cells With Lentiviral Vectors Carrying TBX18 And Induction Of Their Differentiation Into Pacing-like Cells

OBJECTIVE:To observe whether TBX18 lentiviral vector is transfected into adipose-derived stem cells in vitro and can induce its differentiation into pacing-like cells.METHODS:The ADSCs were isolated and cultured by enzymatic digestion method from Sprague Dawley(SD)rat(50g).The cells were observed under light microscope.The cells were randomly divided into lenti-TBX18 group(n=6),lenti-GFP group(n=6)and null group(n=6).The transfected cells were transfected with TBX18 transfection Factor and green fluorescent protein(GFP)lentiviruses,the null group did not transfect the virus.One week later,the expression of related transcription factors TBX3,ISLI,SHOX2,connexin CX45,CX43,CX30.2 and cardiomyocyte specific markers cardiac troponin I(cTNI),actin(a-SMA)protein expression;a-SMA protein expression by immunofluorescence assay.The levels of TBX18 and HCN4 were detected by real-time fluorescence quantitative polymerase chain reaction(RT-PCR)on 7th,14th,21th and 28th day after transfection.RESULTS:After transfection of ADSCs in the experimental group(lenti-TBX18-GFP group),the morphology of the cells was changed and the fluorescence expression was observed by fluorescence microscopy within 4 weeks after transfection.Western blotting showed that the levels of TBX3,ISL1,SHOX2,CX45,CX30.2,cTNl and a-SMA were increased in the experimental group,the expression of CX43 protein was lower than that of the null group.The immunofluorescence of thelenti-TBX18-GFP group detected a-SMA and the lenti-GFP group was negative.RT-PCR results showed that the expression of TBX18 and HCN4 in the lenti-TBX18-GFP was significantly higher than that of the lenti-GFP group on 7th,14th,21th and 28th day after lend virus transfaction(P<0.05).CONCLUSION:TBX18 lentiviral vector is transfected into adipose-derived stem cells in vitro and can be expressed stably in cells,which can differentiate adipose-derived stem cells into pacing cells.