【作者】 王勇；

【导师】 陈虹；

【作者基本信息】 重庆医科大学 ， 内科学， 2012， 硕士

【摘要】 RASGRF2(RAS鸟苷酸释放因子2)是一个富含多结构域蛋白，编码1237个氨基酸，其基因位于人5q13,该区在肺癌中常出现等位基因丢失，因此被认为是一个潜在肿瘤抑制基因。我们前期研究发现RASGRF2在肺癌细胞中存在表达缺失且与异常甲基化有关，在肺癌组织中RASGRF2也存在明显异常甲基化。本课题通过构建RASGRF2-GFP融合基因真核表达载体，转染肺癌细胞并建立稳定表达RASGRF2细胞株，观察过表达RASGRF2对细胞周期的影响，并观察其蛋白的细胞定位，对进一步深入研究RASGRF2基因功能奠定基础。第一部分RASGRF2-GFP融合基因真核表达载体的构建与鉴定。目的：构建带有GFP标签的RASGRF2真核表达载体。方法：将pCMV6-GFP、RASGRF2-pCMV6-Myc-DDK质粒进行SgfI、 NotI双酶切，将RASGRF2基因亚克隆入pCMV6-GFP载体中，构建RASGRF2-pCMV6-GFP重组载体。结果：双酶切与测序证实RASGRF2基因成功克隆到pCMV6-GFP载体中。结论：成功构建了带有GFP标签的RASGRF2真核表达载体，为进一步研究该基因功能提供了工具。第二部分稳定表达RASGRF2肺癌细胞株的建立、细胞定位及功能的初步研究。目的：建立RASGRF2稳定表达的肺癌细胞株、并观察其蛋白细胞定位及其对细胞周期的影响。方法：将pCMV6-GFP、RASGRF2-pCMV6-GFP转染H1299细胞，在倒置荧光显微镜下观察RASGRF2蛋白细胞定位，经G418筛选建立稳定表达RASGRF2肺癌细胞株，经RT-PCR、Western blot检测RASGRF2在稳定细胞株中表达，并将稳定细胞株进行流式检测该基因对细胞周期的影响。结果：RASGRF2-GFP重组蛋白在H1299细胞中成功表达并主要定位于细胞胞质中，稳定细胞株经RT-PCR、Western blot证实RASGRF2在H1299细胞中稳定表达，细胞周期结果显示RASGRF2基因能将细胞周期阻滞在S期。结论：建立了稳定表达RASGRF2基因的肺癌细胞株，对该基因蛋白进行了细胞定位，并发现RASGRF2对细胞周期具有一定的影响，这为进一步深入研究RASGRF2基因在肺癌中的作用奠定了基础。更多还原

【Abstract】 RASGRF2is a rich multi-domain protein, compiled Code of1237amino acids, located on human5q13, allelic loss of the area of lung canceroften is considered a potential tumor suppressor gene. Our preliminary studyfound that RASGRF2loss of expression in lung cancer cells with abnormalmethylation of the relevant RASGRF2,obvious abnormal methylation inlung cancer. the purpose of this study is to construct RASGRF2-GFP fusiongene eukaryotic expression vector, observe the cellular localization ofproteins in lung cancer cells, establish stable expression RASGRF2lungcancer cell strain, and observed influnence on the cell cycle, which lay thefoundation of further study RASGRF2gene function.Part one RASGRF2-GFP fusion gene eukaryotic expression vectorand identification. Objective: to construct RASGRF2eukaryotic expressionvector tagged with GFP.Methods:pCMV6-GFP and RASGRF2-pCMV6-Myc-DDK were digested by SgfI and NotI restriction enzyme, thenRASGRF2was subcloned into the pCMV6-GFP vector to constructRASGRF2-pCMV6-GFP recombinant vector. Results: Enzyme digestion and sequencing data indicated that the recombinant vector was constructedexactly.Conclusion: The RASGRF2eukaryotic expression vector taggedwith GFP constructed successfully，which laid the foundation for furtherstudy RASGRF2gene function.Part two establishment of lung cancer cell strain of stable expressionRASGRF2and cellular localization and function. Objective: to establishstable expression RASGRF2lung cancer cell strain, and observedinflunence on the cell cycle and cellular localization.Methods:lung cancerH1299was transfected with RASGRF2-pCMV6-GFP and observed forlocation of RASGRF2protein under Inverted fluorescence microscope.Thecell strain of stable expressing RASGRF2was established by G418screencing,for flow cytometry cell cycle changes,and was determinedbyRT-PCR and Western blot. Results: The RASGRF2-pCMV6-GFPrecombinant protein was expressed successfully,and located cytoplasm inH1299cells by fluorescence microscope detection.The stable cell strainexpressed stably RASGRF2in H1299cells by RT-PCR, Western blotconfirmed. RASGRF can arrest cell cycle in S phase. Conclusion:estbalishment of the stable expression RASGRF2gene lung cancer cellsstrain, its protein cellular localization, has some influence on the cell cycleand found RASGRF2, which laid the foundation for further studyRASGRF2gene function in lung cancer.更多还原