The Research Of Lentivirus-mediated Gene HCN2 Transfect Bone Mesenchymal Stem Cells

Abstract:Obiective:To study the feasibility of the hyperpolarization-activated cyclic nucleotide-gated cation channel 2 (HCN2) stable transfection in Rat bone marrow mesenchymal stem cells (BMSCs), and to explore the optimal transfection efficiency of HCN2 for the further study of the electrophysiology. It is the basis of the cells’ pacemaker current experimental. Methods:1. BMSCs isolation, culture and identification:BMSCs were isolated and puried by the modified method of whole bone marrow adherent preplating treatment and cultured in DMEM medium with 20% fetal bovine serum. The cells were serial subcultured. The cells were identified by the CD34,CD44,CD45, CD90 through immunohistology and flow cytometer. It was observed the differentiation of BMSCs in the bone-functional properties in vitro bone induction.2. Identify the gene HCN2 amplified and purified by protein electrophoresis and the gene sequence.3. The construction and examination of the recombinant Lentivirus carrier(FIV-HCN2):Recombinant Lentivirus were increased, sublimated and measured of the titer. Taking the EGFP as signal gene ascertains the best MOI to transfect the BMSCs. Observe the change of expression that FIV-EGFP in the BMSCs transfected as the time increasing. The viability was distinguished between FIV-HCN2 transfected BMSCs and non-transfected BMSCs with flow cytometer. Results:1. the modified method of whole bone marrow adherent preplating treatment can obtain the high purity (>90%) and high vigor (>90%) BMSCs. It was showed that the culture cell assumed spindle-shaped, the fusiformate and the polygon by the morphology observation. The CD44 and the CD90 were positive, while the CD34 was negative by the immuno-chemistry dyes. They were conformed to the shape and the superficial mark of the BMSCs; The flow cytometer analysises that the CD44 positive rate of the cells was 99.5% and the CD90 positive rate of the cells was 90.5%. We saw obviously the massive calcium tubercle in the BMSCs induced after the alizarin element dyes. It can explained that the BMSCs has the ability to be induced to the osteoblast.2.The amplify method of the HCN2 plasmid is reliable. The fragment which was from the HCN2 plasmid digested confirned the size of the normal HCN2 gene. The result of the gene sequence agreeed with the GeneBank.3. Construction of the reorganizes Lentiviral-HCN2. We used the virus packed supernate to make the viral titer, we donot see the fluorescence; The BMSCs were infected at the 24h,48h,72h by the supernate, we cannot examine the meaningful green fluorescence.Conclusion:1. We could obtain the high purity and high vigor BMSCs by using the modified method of whole bone marrow adherent preplating treatment。The BMSCs proliferated rapidly in vitro. The number of cells in experiments can be achieved in the short term.2. The amplify method of the HCN2 plasmid is reliable. The fragment which was from the HCN2 plasmid digested confirned the size of the normal HCN2 gene. The result of the gene sequence agreed with the GeneBank.3. Lentiviral HCN2 transfected stem cells needs further study.