贵州医科大学学报

2017, v.42;No.201(06) 626-630

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核不均一核糖核蛋白C2基因克隆及其对肝癌细胞生长的影响
Clone of RNPC2 Gene and its Influence on the Growth of Hepatocellular Carcinoma Cell

孙达权;石松;徐莹莹;雷霆雯;莫晓川;王筑婷;李红梅;徐国强;
SUN Daquan;SHI Song;XU Yingying;LEI Tingwen;MO Xiaochuan;WANG Zhuting;LI Hongmei;XU Guoqiang;Department of Biochemistry and Molecular Biology,Guizhou Medical University;Department of Physiology,Guizhou Medical University;

摘要(Abstract):

目的:探讨人核不均一核糖核蛋白C2(RNPC2)基因在肝癌细胞生长增殖中的作用。方法:采用RTPCR方法扩增肝癌细胞SMMC-7721 RNA中RNPC2基因编码区的目的片段,构建真核表达载体p EGFP-RNPC2并进行细胞转染;采用G418抗生素筛选稳转细胞,SDS-PAGE和Western blot法测定稳转细胞内RNPC2蛋白表达,CCK-8法测定稳转细胞的增殖能力。结果:从培养的肝癌细胞SMMC-7721中抽提总RNA并反转录成c DNA,通过RNPC特异性引物扩增出RNPC2基因;测序鉴定获得双酶切分离纯化目的片段后,插入真核表达载体p EGFP-C1中,成功构建真核表达载体p EGFP-RNPC2;通过细胞稳定转染、G418药物筛选和荧光显微术获得表达外源融合蛋白GFP-RNPC2的稳转细胞株,并用Western blot验证及CCK-8分析证明GFP-RNPC2稳转细胞的生长增殖速率增加。结论:RNPC2可以促进肝癌细胞生长增殖。
Objective: To study the function of human RNPC2 gene on the growth and proliferation of hepatocellular carcinoma cell. Methods: By the method of RT-PCR,the target fragment of RNPC2 gene coding in hepatoma carcinoma cell SMMC-7721 RNA is proliferated to structure eukaryotic expressional vector p EGFP-RNPC2 and conduct cell transfection. RNPC2 expression of protein in stable cell is measured by G418 antibiotic screening stable cell and western blot measuring. Proliferation capacity of stable cell is measured by the method of CCK-8. Results: mRNA,extracted from hepatocellular carcinoma cell SMMC-7721,was reverse transcribed into c DNA,and the CDS region of RNPC2 gene was amplified by PCR with specific RNPC primers. EcoR I and Xho I were explored to separate the goal DNA fragment and be inserted into eukaryotic expressional vector p EGFP-C1 to construct p EGFP-RNPC2. Then,p EGFP-RNPC2 was transfected into SMMC-7721 cell and screened by G418 to harvest GFP-RNPC2 stable cell line which was identified by western blot. At last,the growth and prolifer-ation rate of RNPC2 stable cell increased detected by CCK-8. Conclusion: RNPC2 can promote the growth and proliferation of hepatocellular carcinoma cell.

关键词(KeyWords): 肝肿瘤;核不均一核糖核蛋白C2;细胞;细胞增殖;真核表达;脂质体;转染
liver neoplasms;nuclear heterogeneous ribonucleoprotein C2;cells;cell proliferation;eukaryotic expression;liposomes;transfection

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基金项目(Foundation): 国家自然科学基金(81560390);; 贵州省科学技术基金[黔科合J字(2010)2,黔科合J字(2014)2025号];; 贵阳医学院院基金(院基金合同字第017号)

作者(Author): 孙达权;石松;徐莹莹;雷霆雯;莫晓川;王筑婷;李红梅;徐国强;
SUN Daquan;SHI Song;XU Yingying;LEI Tingwen;MO Xiaochuan;WANG Zhuting;LI Hongmei;XU Guoqiang;Department of Biochemistry and Molecular Biology,Guizhou Medical University;Department of Physiology,Guizhou Medical University;

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DOI: 10.19367/j.cnki.1000-2707.2017.06.003

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