Transfection efficiency of three different lentiviruses with green fluorescent protein gene to transfect rabbit bone marrow mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND:We explore the transfection effects of different lentiviruses on bone marrow mesenchymal stem cells (BMSCs) from the same source in order to screen out appropriate cell transfection tools.OBJECTIVE:To explore the difference in the transfection efficiency of three different lentiviruses carrying green fluorescent protein (GFP) to transfect the rabbit BMSCs.METHODS:Bone marrow samples were extracted from the ilium of 2-week-old Japan white rabbits,and then the BMSCs were separated,purified and extended using density gradient centrifugation.Cell surface markers were identified using flow cytometry.pTomo-GFP lentivirus,Ad-GFP lentivirus and lenti-GFP lentivirus were used to transfect the BMSCs,and the transfection efficiency was tested using flow cytometry.The cell viability was detected using cell counting kit-8 before and after transfection.RESULTS AND CONCLUSION:As the positive rates of CD44,CD34 and CD45 were 75.64％,6.14％ and 9.48％,respectively,the cultured cells were identified as BMSCs by the flow cytometry.The expression levels of GFP after transfection by pTomo-GFP,Ad-GFP and ienti-GFP were 2.64％,16.72％ and 45.24％,respectively.The cell growth curve showed that pTomo-GFP lentivirus,Ad-GFP and ienti-GFP lentivirus showed certain effects on cell growth,pTomo-GFP and Ad-GFP had an effect on cell growth at incubation period and logarithmic growth phase at 1-8 days after transfection,which may cause some interference to the viability of early cells,ienti-GFP had little effect on cell growth in the short term,but there was a change in cell growth at day 12 after lenti-GFP transfection,and a long-term effect of lenti-GFP on cell viability was considered.Therefore,lenti-GFP lentivirus could highly transfect the BMSCs and could not impact the normal growth of BMSCs,indicating that lenti-GFP lentivirus is suitable for BMSCs tracing and gene transfection.