线粒体融合基因2mfn2在骨肉瘤增殖和凋亡中的功能及其分子机制分析-function and molecular mechanism analysis of mitochondrial fusion gene 2 mfn 2 in proliferation and apoptosis of osteosarcoma.docxVIP

三峡大学硕士学位论文 三 峡 大 学 硕 士 学 位 论 文 PAGE IV PAGE IV 万方数据 Abstract Objective: Mitofusin-2 is a important member of mitofusin family, which has a low expression level in many cancer cells including MG63 osteosarcoma cell. The objective of this study is to observe the impact of Mfn2 on the proliferation and apoptosis of osteosarcoma cell in vivo and in vitro experiments, to further reveal the function of this gene on proliferation and apoptosis of cancer cells, which may lay a solid theory foundation for the treatment of osteosarcoma. Methods: (1) To analysis the expression level of mfn2 in osteosarcoma and para-carcinoma tissue through immunohistochemical technique; to detect the mRNA and protein expression levels of mfn2 in normal and MG63 osteosarcoma cell lines. (2) To construct a pIRES-flag-Mfn2 recombinant plasmid, and then transfect the recombinant plasmid into MG63 cell, and then further screen out clones those expression flag-mfn2. (3) To detect the proliferation and apoptosis of MG63 osteosarcoma cell lines after the expression level of mfn2 has up-regulation: to detect the proliferation through MTT; to observe the cell morphology through inverted fluorescence microscope or electron microscopy; to detect the cell cycle through flow cytometry and to detect apoptosis related protein such as PARP, caspase3; DNA fragmentation detection. (4) To culture the cells those express flag-Mfn2 and Harvest the cell lysates, to purify the flag-Mfn2 protein through Immunoprecipitation and make mass spectrum analysis, and then discovery the interactional protein that proliferation and apoptosis related; to research and analysis the possible mechanism that the mfn2 slows down the proliferation and accelerates the apoptosis of osteosarcoma cell. Results:(1) the expression level of mfn2 in osteosarcoma is significant lower than the normal cell and tissue, and the expression level of mfn2 in osteosarcoma is lower than para-carcinoma tissue. (2) successfully constructe