重组腺病毒载体介导人骨形成蛋白BMP2-15诱导干细胞C2C12成骨作用的研究

发布时间：2018-04-02 01:32

本文选题：骨形成蛋白BMP2-15　切入点：重组腺病毒　出处：《重庆医科大学》2011年硕士论文

【摘要】：目的:以HEK293细胞作为腺病毒包装细胞,采用乒乓感染的病毒复制法,制备表达骨形成蛋白BMP2-15的重组腺病毒,并进一步进行病毒滴度测定与功能鉴定,为第二部分重组腺病毒载体介导人骨形成蛋白BMP2-15诱导干细胞C2C12成骨作用的研究奠定基础。方法:通过乒乓感染、细胞冻融的腺病毒复制法,制备表达骨形成蛋白BMP2-15的重组腺病毒;以荧光表达强度比较法测定病毒滴度;以RT-PCR的方法鉴定制备的表达骨形成蛋白BMP2-15的重组腺病毒的表达功能。结果:(1)获得高滴度的表达骨形成蛋白BMP2-15的重组腺病毒。(2)以高滴度的表达骨形成蛋白BMP2-15的重组腺病毒感染实验细胞株C2C12后,RT-PCR检测结果证明能有效表达目的基因骨形成蛋白BMP2-15。结论:成功制备高滴度的表达骨形成蛋白BMP2-15的重组腺病毒,用该病毒感染实验细胞株,能有效表达目的基因,为进一步的功能实验奠定了基础。第二部分重组腺病毒载体介导人骨形成蛋BMP2-15诱导干细胞C2C12成骨作用的研究目的:以表达骨形成蛋白BMP2-15的重组腺病毒感染C2C12细胞,诱导其向成骨方向分化,通过体外体内实验系统分析所有BMP成骨作用的差异。方法:(1)以表达骨形成蛋白BMP2-15的重组腺病毒感染具有多向分化潜能的干细胞C2C12,碱性磷酸酶活性测定检测BMP2-15诱导C2C12细胞向骨细胞分化的功能强弱。(2)目的BMP基因诱导C2C12细胞后,注射入免疫缺陷鼠体内,通过体内成骨的实验研究比较不同BMP成骨作用的差异。结果:(1)筛选出进一步实验的目的基因BMP2、4、6、7、9。(2)目的BMP基因诱导C2C12细胞成骨后,体外体内实验证明除传统BMP2及BMP7外,BMP9具有更强的成骨作用。结论:以表达骨形成蛋白BMP2-15的重组腺病毒感染具有多向分化潜能的干细胞C2C12,碱性磷酸酶活性测定检测BMP2-15诱导C2C12细胞向骨细胞分化的功能强弱,筛选出具有诱导成骨分化功能的目的基因BMP2、4、6、7、9,进一步通过体外体内实验证明除传统BMP2及BMP7外, BMP9具有更强的成骨作用。
[Abstract]:Objective: to prepare recombinant adenovirus expressing bone morphogenetic protein (BMP2-15) by ping-pong virus replication using HEK293 cells as adenovirus packaging cells, and to determine the titer and function of the recombinant adenovirus. The second part of the recombinant adenovirus vector mediated osteogenesis of human bone morphogenetic protein BMP2-15 induced stem cell C2C12 osteogenesis. Methods: recombinant adenovirus expressing bone morphogenetic protein (BMP2-15) was prepared by ping-pong infection and freeze-thaw adenovirus replication. The expression function of recombinant adenovirus expressing bone morphogenetic protein BMP2-15 was identified by RT-PCR. Results the recombinant adenovirus with high titer of bone morphogenetic protein BMP2-15 was obtained. The recombinant adenovirus expressing bone morphogenetic protein BMP2-15 with high titer was infected with C2C12. The results of RT-PCR showed that the recombinant adenovirus expressing bone morphogenetic protein BMP2-15 could express the target gene BMP2-15 effectively. Conclusion: the recombinant adenovirus expressing bone morphogenetic protein BMP2-15 with high titer was successfully prepared. The recombinant adenovirus infected with the recombinant adenovirus could effectively express the target gene and lay a foundation for further functional experiments. The second part of the study on the osteogenesis of stem cell C2C12 induced by recombinant adenovirus vector mediated human bone formation egg BMP2-15. Aim: to infect C2C12 cells with recombinant adenovirus expressing bone morphogenetic protein (BMP2-15) and induce them to differentiate into osteoblasts, and to analyze the difference of osteogenesis of all BMP cells in vitro and in vivo. Methods the recombinant adenovirus expressing bone morphogenetic protein (BMP2-15) was used to infect C2C12 stem cells with multidirectional differentiation potential. Alkaline phosphatase activity assay was used to detect the function of BMP2-15 in inducing C2C12 cells to differentiate into bone cells. The osteogenic effects of different BMP were compared by in vivo osteogenesis in immunodeficient mice. Results (1) the target gene BMP2, BMP2, was selected to induce the osteogenesis of C2C12 cells. The in vitro experiments showed that BMP9 had a stronger osteogenic effect in addition to traditional BMP2 and BMP7. Conclusion: C2C12 stem cells with multiple differentiation potential were infected with recombinant adenovirus expressing bone morphogenetic protein (BMP2-15). Alkaline phosphatase activity was used to detect the function of BMP2-15 in inducing C2C12 cells to differentiate into bone cells. The target gene BMP2O4, BMP2, was screened out, and the in vitro experiments proved that BMP9 had stronger osteogenic effect than the traditional BMP2 and BMP7.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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