Expression Of Liposomes-mediated BMP2 And VEGF165 Double Gene Modified BMSCs

Objective:Through the way of whole marrow culture to separate and purify the bone marrow stromal stem cells (BMSCs) from mice. Construct the recombinant pYr-adshuttle-4 plasmid which is carrying BMP2 and VEGF165, and transfect it into bone marrow stromal cells by liposome, then culture the transfected BMSCs in vitro to observe both the biological characteristics and the ability of differentiating osteoblast, and discuss the differences between double gene-modified and single gene-modified rat bone marrow stromal cells by BMP2 and VEGF165. Method:Get the bone marrow from bilateral femur of mice, and use the whole bone marrow culture to train originally, to separate and purify the cultured bone marrow stromal cells until the third generation by changed liquid and passages, and use it for following experiment. Construct the recombinant pYr-adshuttle-4 plasmid which is carrying bone BMP2 and VEGF165, and transfect it into bone marrow stromal cells by cationic liposome, and the transfected cells are divided into pYr-adshuttle-4-BMP2 group, pYr-adshuttle-4-VEGF 165 group, pYr-adshuttle-4-BMP2, pYr-adshuttle-4-VEGF165 group, and empty vector group.Moreover, observe the biological characteristics and the ability of bone induction. After the transfection,observe it for 7days. Then test the content of mRNA in BMP2 and VEGF165 in 48hs Western-Blot tested the protein expression of BMP2 and VEGF165. After a week the transfection cells were detected osteogenic ability of each group-Base phosphatase. Results:Bone marrow stromal cells are isolated, separated and purified successfully from mice, and they show long spindle sample stick wall growth. Success to construct the recombinant pYr-adshuttle-4 plasmid which is carrying BMP2 and VEGF165, and success to transfect the recombinant pYr-adshuttle-4 plasmid into BMSCs by liposomes-mediated gene transfer method, and it is high transfected rate, no cytotoxicity, and the vitality of cell growth is enhanced. RT-PCR detection displayed that the mRNA level of BMP-2+VEGF165 double gene expression group is higher than BMP-2,VEGF165 single gene expression group. Western Blot test showed that the level of expression quantity of BMP-2 and VEGF165 genome protein is significantly greater than single genome. The ALP activity in double group bone marrow stromal cells is obviously higher than each single group. Conclusion:1.Whole bone marrow culture can success to derive BMSCs from mice.2.The liposomes-mediated gene transfer method has high efficiency transfected rate in vitro, the transfected cells has low toxicity. The method makes highly stable common expression BMP-2 and VEGF-165 gene, and induced to osteoblast differentiation. 3.BMP2,VEGF165 double gene modify BMSCs have high ability than single gene modify BMSCs to induced osteoblast differentiation.