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pEGFP-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cD-NA,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pEGFP-C1中,成功构建真核表达载体pEGFP-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 pEGFP-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 cDNA,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 pEGFP-C1 to construct pEGFP-RNPC2.Then,pEGFP-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 proliferation rate of RNPC2 stable cell increased detected by CCK-8.Conclusion:RNPC2 can promote the growth and proliferation of hepatocellular carcinoma cell.
展开▼
