Biological Characteristics Of Lentivirus-mediated HBMP-2 And HVEGF-165 Double Gene Transfection Into BMSCs

Objective: To verify the feasibility of lentivirus-mediated transfection of hBMP-2 and hVEGF-165(two transfections)of BMSCs and better induction of osteogenic performance of BMSCs after transfection.Methods: BMSCs were isolated and extracted by flow cytometry.The lentivirus carrying hBMP-2 gene was transfected into the third generation of rabbit BMSCs.After doubling the cells,half of them were used as single gene transfection group,and half of them used lentivirus carrying hVEGF-165 gene.Transfection was again performed and observed under a fluorescence microscope.So far,BMSCs were divided into 4 groups,group A was untransfected group,group B was twice empty transfected with fluorescent protein gene,group C was BMP2 single gene transfection group,group D was hBMP-2 and hVEGF-The 165 double gene transfection group.The expression and protein of target protein were detected by Western Blot at different time points after transfection.The proliferation activity of each group was detected by MTT.The alkaline phosphatase activity of each group was detected 14 days after transfection.After transfection and 6 weeks osteogenic induction,alizarin red staining was used to detect bone nodules.Results: the expression of green and red fluorescent protein was observed in the 1.B,C and D groups.After transfection,3D C,D group has the corresponding protein expression after transfection,7d,14 d,21d,protein detection had no significant change(p>0.05)2.;cell proliferation was detected in the group D proliferation slightly faster than C group(p<0.05),C group was significantly faster than A and B group(p<0.05),go A,B group cell proliferation curve direction were similar(p>0.05);3.A B group,alkaline phosphatase staining only about 5% cells were deeply stained,90% in C group,D group was 80%.C,D group showed significantly higher ALP activity than A,B group,D group was higher than that of group C was about 1/5;The number of bone nodules stained by alizarin red in group C was significantly higher than that in group A and B.The number of bone nodules in group D was slightly larger and larger than that in group C.Conclusions:adenovirus mediated hBMP-2 and hVEGF-165 gene transfection can be successfully transferred by two times in BMSCs,and the long-term stable expression.The expression of double factor by stimulating cell alkaline phosphatase produced better promote cell the osteoblastic differentiation.