硒蛋白PP1(SEPP1)过表达抑制786-O和769-P人肾癌细胞增殖并引起G2/M期阻滞  

Overexpression of SEPP1 inhibits the proliferation and induces cell cycle G2/M arrest of 786-O and 769-P human renal carcinoma cells

作　　者：刘侃[1] 赵超飞 陈建文[1] 巫胜攀 姚远新 武翀 罗国雄[1] 张旭[1] 

机构地区：[1]解放军总医院泌尿外科,北京100853

出　　处：《细胞与分子免疫学杂志》2016年第6期764-769,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高技术研究发展计划(863)(2014AA020607)

摘　　要：目的构建硒蛋白PP1(SEPP1)基因的慢病毒质粒并研究SEPP1对人肾透明细胞癌细胞增殖的影响。方法以HEK293T细胞的c DNA为模板,通过PCR法获得人SEPP1全长序列片段,并与慢病毒表达载体p LV-EGFP(2A)Puro质粒相连接,获得重组p LV-EGFP(2A)Puro-SEPP1载体并进行基因测序验证。将重组慢病毒载体与包装质粒共同转染HEK293T细胞,进行病毒包装。用病毒上清感染786-O和769-P细胞,采用Western blot法检测SEPP1蛋白表达。按照重组慢病毒感染细胞、空载体感染细胞、未感染细胞进行分组,通过MTS法检测细胞增殖活性、细胞平板克隆形成实验检测克隆形成能力、流式细胞术检测细胞周期。结果酶切后凝胶电泳得到与SEPP1的DNA大小相一致的片段与空载体片段,基因测序的结果与SEPP1序列完全一致。经p LV-EGFP(2A)Puro-SEPP1感染的786-O、769-P细胞在感染48 h后,荧光显微镜下可见EGFP明显表达,实验组与空载体组、空白对照组相比较,SEPP1蛋白表达水平均明显提高。p LV-EGFP(2A)Puro-SEPP1感染组的细胞增殖能力降低,SEPP1过表达细胞集落形成能力明显降低,SEPP1过表达引起肾癌细胞G2/M期阻滞。结论在786-O和769-P细胞过表达SEPP1显著降低细胞增殖能力,并在786-O细胞中引起G2/M期阻滞。Objective To establish selenoprotein P,plasma 1（ SEPP1） gene recombinant lentiviral vector and investigate the effect of SEPP1 on the proliferation of human clear cell renal cell carcinoma（ ccRCC） cells. Methods c DNA sequence of SEPP1 was cloned from the total c DNA of HEK293 T cells by PCR. Then,the c DNA fragment was combined with the p LV-EGFP（ 2A） Puro vector and the constructed plasmid p LV-EGFP（ 2A） Puro-SEPP1 was transfected into HEK293 T cells for packaging the virus. Forty-eight hours after transfected with the virus supernatant,the level of SEPP1 protein in 769-P and786-O cells were tested by Western blotting. Cells were divided into recombinant lentivirus-infected cells,empty vector lentivirus-infected cells and the blank control cells. Cell proliferation rate was detected by MTS assay,colony forming ability was evaluated by plate clony formation assay and cell cycle change was assayed by flow cytometry after transfected with p LV-EGFP（ 2A） Puro-SEPP1 or empty p LV-EGFP（ 2A） Puro vector. Results Enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid p LV-EGFP（ 2A） Puro-SEPP1 was constructed successfully. After being infected by the virus supernatant,the 786-O and 769-P cells expressed EGFP. Compared with the empty vector group and the blank control group,expression level of SEPP1 in the experimental group was much higher. The cell proliferative ability was inhibited in the cells overexpressing SEPP1,and the colony forming ability of SEPP1-overexpressed cells evidently decreased. Cell cycle was arrested in G2/M phase in 786-O cells overexpressing SEPP1. Conclusion The recombinant plasmid p LV-EGFP（ 2A）Puro-SEPP1 has been constructed successfully. Overexpression of SEPP1 could significantly reduce the proliferation rate of786-O and 769 P cells,and cause G2/M phase arrest of 786-O cells.

关 键 词：硒蛋白PP1(SEPP1) 重组慢病毒载体 肾透明细胞癌 肿瘤增殖 

分 类 号：R737.11[医药卫生—肿瘤] R392-33[医药卫生—临床医学]