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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 construct a lentiviral plasmid containing the selin protein PP1 (SEPP1) gene and study the effect of SEPP1 on the proliferation of human renal clear cell carcinoma cells. Methods The full-length human SEPP1 fragment was obtained by PCR using c DNA of HEK293T cells as a template. The full-length cDNA of human SEPP1 was ligated with the lentiviral vector p LV-EGFP (2A) Puro plasmid to obtain the recombinant p LV-EGFP (2A) SEPP1 vector and gene sequencing verification. The recombinant lentiviral vector and packaging plasmid co-transfected HEK293T cells for viral packaging. 786-O and 769-P cells were infected with virus supernatant and the expression of SEPP1 protein was detected by Western blot. According to the recombinant lentivirus-infected cells, the empty vector was infected, uninfected cells were grouped, the cell proliferation activity was detected by MTS assay, the clonogenic capacity was detected by cell plate clone formation assay, and the cell cycle was detected by flow cytometry. Results After restriction enzyme digestion gel electrophoresis, the fragment and empty vector fragment consistent with the DNA size of SEPP1 were obtained. The result of gene sequencing was exactly the same as that of SEPP1. The expression of EGFP in 786-O and 769-P cells infected by pVEGF-Purp-SEPP1 was observed under fluorescence microscope 48 hours after infection. Compared with the blank control group and the blank control group, the expression of SEPP1 protein Expression levels were significantly improved. p-EGFP (2A) Puro-SEPP1 infected cells decreased the ability of cell proliferation, SEPP1 over-expression of colony-forming ability was significantly reduced, over-expression of SEPP1 cells caused G2 / M phase arrest. Conclusion Overexpression of SEPP1 in 786-O and 769-P cells significantly reduced cell proliferation and caused G2 / M arrest in 786-O cells.