【作者】 马雷；

【导师】 姚华；

【作者基本信息】 新疆医科大学 ， 劳动卫生与环境卫生学， 2010， 博士

【摘要】 目的：骨缺损畸形是颌面外科、骨科、整形外科临床常见病。颌骨缺损的修复问题一直是口腔颌面外科较难解决的问题,现常规的修复方法很多,但各有其优缺点。组织工程化骨是利用生物组织工程技术将种子细胞与支架材料复合,形成具有与自体骨结构功能相似的骨组织,它弥补了多种单一植骨材料的缺陷,结合了它们各自的优点,具有组织相容性好、骨诱导能力强、取材方便、可快速修复缺损等特性。用组织工程方法构建种子细胞、支架材料和生长因子复合的生物活性人工骨被认为是未来进行骨缺损修复和骨再造最有效的方法之一。通过人碱性成纤维生长因子(basic fibroblant growth factor, bFGF)转染以增加组织细胞bFGF的分泌量,提高bFGF蛋白在生物体骨缺损局部组织中的浓度,达到促进骨愈合的目的。但是,如何提高质粒对于靶组织的转染效率、寻求一种高效、简便的转染手段,如何缩短鉴定转染是否成功的时间,以及在基因转染促进骨愈合的过程中,一直未得到很好的阐明。本研究目的包括以下几个方面：1)建立稳定的、基因序列正确的、检测方便的、和GFP-慢病毒载体,并检测转染293-FT细胞的效率及转染后的生物学活性；2)获得骨髓间充质干细胞(Bone mesenchymal stem cells, BMSCs),bFGF-慢病毒载体转染诱导第3代的犬BMSCs,了解该质粒转染犬骨髓间充质干细胞的效率及转染后的生物学活性；3)体内观察对比bFGF基因修饰rMSCs细胞活性的影响以及诱导异位成骨的情况,为构建更优化的骨组织工程种子细胞和组织工程化骨提供重要的实验基础。方法：1)bFGF基因真核表达载体的构建设计人bFGF基因引物,用Trizol法提取胎盘组织总RNA,用逆转录聚合酶链反应(RT-PCR)的方法扩增bFGF基因,连接至ViraPowerTM慢病毒载体系统,经Xho-Ⅰ, BamH-Ⅰ双酶切和测序并与Genebank中序列进行比较分析。2)bFGF基因遗传修饰的犬骨髓间充质干细胞的建立：①结合Percoll分离液密度梯度离心和差速贴壁传代筛选法相,分离培养犬骨髓间充质干细胞。②在脂质体转染剂Lipofectamine2000的介导下,bFGF-pLenti6/V5-D-TOPO表达质粒同包装质粒pLP1、pLP2、包膜质粒pLP/VSVG共转染293FT细胞株,收集bFGF-慢病毒上清转染诱导后第3代的犬BMSCs, Blasticidin压力筛选稳定表达株。③设计GFP基因引物,以GFP-PMSLV-Plazmid为—1—模板,采用PCR的方法扩增GFP基因,连接至pLenti6/V5-D-TOPO(?)表达质粒,GFP-慢病毒载体的构建及转染BMSCs过程同bFGF-慢病毒。RT-PCR方法检测bFGF mRNA水平的表达。④RT-PCR检测转染组细胞中目的基因mRNA表达情况,Western blot,细胞免疫化学检测各转染组细胞胞浆中目的蛋白表达情况；ELISA检测各各转染组细胞培养上清中目的蛋白的分泌情况。3)bFGF基因转染的rMSCs成骨活性的体内实验研究实验共分为4组进行：A、对照组(空白组)；B、p-磷酸三钙组；C、MSCs细胞复合β-磷酸三钙组；D、bFGF基因修饰rMSCs+β-磷酸三钙组；①在体外分别将rMSCs和bFGF基因修饰rMSCs接种于多孔支架材料磷酸三钙上。②将不同组细胞／材料复合物植入犬牙槽骨内。③每组分别于植入3月后取材,石蜡包埋,组织病理学切片,HE染色分析新骨形成情况；④术后4、8和12周进行X光片、4、8周CT摄片观察骨缺损变化、骨皮质及β-磷酸三钙降解情况。结果：1)RT-PCR自脑组织cDNA库中扩增出bFGF基因；成功将bFGF基因克隆入真核表达载体,构建了bFGF-pLenti6/V5-D-TOPO表达质粒,DNA测序证明,所克隆目的基因序列与Genebank收录一致。2)bFGF真核表达质粒转染了犬骨髓间充质干细胞后,经抗生素Blasticidin压力筛选获得了具有抗性的细胞克隆；GFP真核表达质粒转染了犬骨髓间充质干细胞48小时后,倒置荧光显微镜下显示转染后的犬骨髓间充质干细胞内可见绿色荧光蛋白分布于整个细胞,呈胞浆分布。培养第八代,转染的细胞依然存活,绿色荧光蛋白持续表达；RT-PCR结果显示抗性细胞克隆中均有大量目的基因bFGFmRNA转录,ELISA检测培养上清中目的蛋白表达阳性,Western blot结果显示胞浆中有目的蛋白的表达。3)犬牙槽骨缺损异位诱导成骨实验显示：基因修饰MSCs组与单纯MSCs对照组相比,材料降解和替代的速度明显提高,有较多的新骨形成；实验动物牙槽骨骨缺损愈合过程的X线4周、8周、12周观察显示及4、12周的CT扫描,bFGF基因修饰rMSCs+β-磷酸三钙组骨缺损在骨皮质的连续性、p-磷酸三钙的降解速度及骨缺损愈合的大小优于MSCs细胞复合p-磷酸三钙组,p-磷酸三钙组明显优于对照组,bFGF修饰的MSCs组在各阶段均有比其他组更明显的材料替换速度。结论：1)成功构建了携带人bFGF和GFP基因的真核表达载体。2)含有人bFGF基因序列可以通过脂质体介导有效地导入犬MSCs中并得到表达。3)人bFGF基因对犬骨髓间充质干细胞修饰转染并能在骨髓间充质干细胞中翻译、表达产生bFGF基因。4)bFGF能够启动或诱导MSCs成骨方向分化和能够促进新生血管形成,对rMSCs进行修饰后,能够在犬牙槽骨内有效地促进异位骨的形成。更多还原

【Abstract】 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.更多还原