bfgf基因修饰的mscs细胞复合β磷酸三钙修复骨缺损实验分析word格式论文.docx

新疆医科大学医学博士学位论文with bFGF gene. ①MTT method was used to analyze the cell proliferation of differentgroups. ②We tested the alkaline phosphatuse (ALP) activity in endochylema. ③The concentration of osteocalin (OCN) which was secreted into cell culture by MSCs modified with different genes was tested by radioimmunity method. 4) The investigation of osteogenous capacity of rMSCs modified with bFGF gene in vivo. We divided those cells into 4 groups: A.contrast group; Bβ-TCP; C.MSCsβ-TCP; D.rMSCs+β-TCPmodified with bFGF gene. ①We seeded the different group cells on the β-TCP scaffoldin vitro. ②The cells/scaffold composites were implanted into mandible of Beagle. ③ The samples were taken at 3 methods post-implian.We used pathological and histology to analyze bone and vessel formation. Rsults: We obtained bFGF gene from brain cDNA library. bFGF gene was cloned into Lipofectamine2000. The eukaryotic expression plasmids, bFGF-plentib, VS-D-TOPO, were constructed, and the results of DNA sequencing showed that the gene sequences we have cloned were identical with those in GeneBank. We thrafected MSCs with eukaryotic expression plasmids. After selected with antibrotin Blasticidin, we got some cell clones which can survive in DMED mediumsupplied with fatal concentration. RT-PCR showed the mass transcription of BMP-2 and bFGF mRNA in tranfected MSCs. Western blot, immunocytochemistry and ELISA confirmed the expression of exogenous gene in tranfected cells and cell culture medium. After to have been transfected with bFGF gene, MSCs got an increased proliferation ability. The in vitro experiment results showed that: the allalire phosphatase (ALP) activity can be downregulated, but the secretion of osteocalcin (OCN) of MSCs can be improved by bFGF gene modified. Ecotopic bone formation experiment: Compared with the control group, the degradation speed of scaffold is more rapid in each of gene. Modified MSCs group, and there are new bone formation in each of gene modified MSCs g