An Expermental Study Of MSCs And β-TCP Restoration Of Mandible Defect Which Modified By BFGF Gene

Objective:Elevating the concentration of the bFGF protein at the bone defect site by tranfering the bFGF into the issues around the defect site. But how to improve the efficiency of gene transfection, shorten the verifying time of a plasmid transfection, find a high efficient and easier method to transfer a plasmidis still unclear. We develop this study aim to:1) contruct the stable and easily identified bFGF and GFP.2) Bone mesenchymal stem cells (BMSCs) from the dog.With the promotion of Lipofectamine 2000, bFGF-plentib/V5plasmid and packaging plasmid pLP1, pLP2, plP/vsvG transfected 293FT cell line, bFGF-lentivirus supernatant was collected and infected the passageⅢBMSCs.3) Oberve rMSCs transfected the gene of bFGF and compare the osteogenous capacity in vitro. Methods:1) Construction of expressing carrier by bFGF gene. bFGF gene primers were designed, total RNA was extracted from placental tissue using TRIZOL.The hbFGF gene amplified by RT-RCR, and the PCR product was connected to the Viro Power TM lentivirus carrier system, which compared and analysed with Genebank srquencing by the Xho-Ⅰand Bam H-Ⅰdouble digestion and sequencing.2) The establishment of bone marrow mesenchymal cells modified by bFGF gene.①Dog MSCs were isolated by combination means of the Percoll intensity gradient centrifugation method and passaged screening were cultured in DMEM medium supplemented with 10%fetal bovine serum.②With the promotion of Lipofectamine 2000, bFGF-plentib/V5plasmid and packaging plasmid pLP1, pLP2, plP/vsvG transfected 293FT cell line, bFGF-lentivirus supernatant was collected and infected the passageⅢBMSCs.③The GFP gene is umplified by PCR from GFP-PMSLV-Plazmid, GFP gene was connected to plentib\V5SCs.The bFGF and mRNA was detected by RT-PCR.④The mRNA expression of exogenous gene in rMSCs was detected by RT-PCR; The intron cell target protein expression was detected by Western blot, immunocytichemistry; ELISA was used to analyze the secretion of target protein.3) The study of bioactivity of MSCs modified with bFGF gene. We tested those cells in 2 groups:A MSCs; B MSCs modified with bFGF gene.①MTT method was used to analyze the cell proliferation of different groups.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; B(3-TCP; C.MSCsβ-TCP; D.rMSCs+β-TCP modified with bFGF gene.①We seeded the different group cells on the (3-TCP scaffold in vitro.②The cells/scaffold composites were implanted into mandible of Beagle.③The 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 medium supplied 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 group. After 1 month of operation, there were a lot of osteoblast like cells and chondrocyte at the edge of scaffold, cell matrix accumulated. Some new born capillary began to form and expand into scaffold. bFGF gene modification group showed a more obvious scaffold substitute speed than the other groups. Most part of scaffold was absorbed at the end of several months after implanta-tion. The relative bone area and capillary density of these two groups are bigger than the other groups. Conclusion:To construct eukaryotic expression plasmids carrying human bFGF gene and GFP gene successfully. Human bFGF gene can be transfected into dMSCs mediated by liposome and be expressed successfully. Human bFGF gene transfected by dog BMSCs shows persistent expression of GFP as a manner of endochylema distribution in the observation of the fluorescencet inverted microscope. MSCs transfected with bone induced and capillary induced gene can improve the ectoptic bone formation ability and improve the vascularization of new bone.