Empirical Study Of SVV-Lentivirus Transfect With MSCs In Vitro

Objective:The limited marrow mesenchymal stem cells viability after transplantation in vivo have restricted their regenerative capacity. In our study, we genetically modified MSCs with an antiapoptotic Survivin gene in vitro and evaluated cell survival and functional improvement, to find a new back up for stem cells of the antiapoptotic.Methods:Isolating C57BL / 6 mouse bone marrow cells, MSCs by whole bone marrow adherence method was adopted, take the Passage 11 for next trial. Firstly: charted the growth curves of the not transfection passage MSCs, the specific surface markers SCA-1,CD29,CD34,CD44 and CD117 were detected by flow cytometry (FCM), Osteogenic and adipogenic induction was performed on MSCs, expression of survivin mRNA in MSCs determined by rt-PCR. Secondly, prepared compound DNA-Lipofectamine 2000, transfeced to 293FT and collected culture solution with virus, make Svv-lentivirus and control lentivirus, determine virus titer; Svv-lentivirus and control lentivirus transfect with MSCs separately and when transfection efficiency near 90% count the MOI; Osteogenic and adipogenic induction were performed on MSCs; comparing the survivin quantity of anterior-posterior by rt-PCR.Results:The cells displayed fusiform shape as fibroblasts, the surface highly express SCA-1, CD29, CD34, CD44, lowly express CD117; the growth curves showed that cells grew slowly in the first three days, then growth accelerated and reached the top at the seventh day; after 20 days of Osteogenic induction, alizarin red stain showed that alizarin red was positive in cells, and after 14 days adipogenic induction, oil red O stain showed that lipide sediment existed; survivin mRNA was positive in MSCs by rt-PCR. Svv-lentivirus and control lentivirus were packaged successfully, virus titer was about 7.1×10~7 TU/ml and control was 8.3×10~7 TU/ml ;when transfection efficiency near 90% , the MOI of Svv-lentivirus and control lentivirus were 28.4 and 8.3 separately; after transfection the MSCs still have the capability of Osteogenic; survivin mRNA was over express in MSCs after transfection by rt-PCR.Conclusion: MSCs can be cultured abundantly by whole bone morrow adherence method, and expression of survivin is positive, and indicating that survivin may participate in the anti-apoptotic of MSCs; after transfection the MSCs still have the capability of differentiation and over express survivin, to provide the basis of researching the survival time in vivo.