CENP－I表达下调对HEK293细胞生长及染色体分离的影响  

Down-regulated centromere protein-I arrests cell growth at G_2/M phase in human embryo kidney cells

作　　者：袁泰先[1] 蔡燕[1] 彭乙华[2] 翁亚光[1] 施琼[1] 刘子杰[1] 刘斌[1] 李素彦[1] 

机构地区：[1]重庆医科大学临床检验诊断学省部共建教育部实验室、重庆市市级重点实验室,重庆400016 [2]川北医学院附属医院急诊科,四川南充637000

出　　处：《第三军医大学学报》2009年第16期1569-1572,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30371485);教育部博士培养基金(20060631012);重庆市重点项目[渝科发计字(2004)47号]~~

摘　　要：目的构建CENP-I特异性RNA干扰真核表达载体,体外观察抑制CENP-I基因表达对HEK293细胞生长、细胞周期和染色体数目异常率的影响。方法构建CENP-IsiRNA真核表达载体,脂质体法将pGenesil-1空载体和pGene-sil-1/CENP-I-siRNA-1、pGenesil-1/CENP-I-siRNA-2、pGenesil-1/CENP-I-siRNA-3真核表达载体分别导入人胚肾HEK293细胞。转染后24、48、72h收集细胞用Western blot和FQ-PCR检测HEK293细胞内CENP-I蛋白及mRNA水平的表达情况,以确定干扰效果及最佳干扰时间。MTT法检测各实验组细胞生长情况,流式细胞术检测各组细胞的细胞周期分布,Giemsa染色法计数细胞分裂指数,常规法制备染色体标本。结果成功构建CENP-I siRNA真核表达载体,筛选出具有干扰效果的质粒载体即pGenesil-1/CENP-I-siRNA-3,转染人胚肾HEK293细胞72h后可使CENP-I蛋白及mRNA表达显著下凋。CENP-I表达降低的HEK293细胞生长速度明显减慢(P<0.05),G2/M期细胞增加,分裂期细胞比例增大。结论RNA干扰有效地特异性抑制着丝粒特异性蛋白质CENP-I的表达。CENP-I的抑制可使人胚肾HEK293细胞生长减慢,增殖能力减弱,使细胞分裂期延长。Objective To construct the RNA interference eukaryotic expression vector targeting human centromere protein-I （CENP-I） and to observe its effect on the growth of human embryo kidney 293 ceils （HEK 293）. Methods The expression vectors of pGenesil-1/CENP-I-siRNA-1, pGenesil-1/CENP-I-siRNA-2 and pGenesil-1/CENP-I-siRNA-3 were constructed by gene recombination and then were transfected into the HEK293 cells by liposome. The expressions of CENP-I at the protein and mRNA levels were detected by Western blotting and fluorescence quality PCR （FQ-PCR）. The effective vector and the best transfection time were selected. The growth and the cell cycle of the transfected cells were assessed by MTT assay and flow cytometry. Giemsa was used to stain the transfected cells to calculate the mitotic index. Results Sequence-specific siRNAs targeting CENP-I significantly down-regulated the expression of CENP-I in HEK293 cells. The recombinant plasmid of pGenesil-1/CENP-I-siRNA-3 was the effective vector. After transfecting for 72 h the best inhibited efficiency was achieved. In CENP-I-siRNA transfected cells, the rate of cell growth was decreased markedly. Cells at G2/M phase and the mitotic index were increased conspicuous compared with the cells transfected with the blank vector or untransfected. Conclusion Down-regulation of CENP-I in HEK293 cells by sequence specific siRNA delays the cell growth and postpones the cell division.

关 键 词：CENP—I基因 RNA干扰 细胞分裂指数 

分 类 号：R322.61[医药卫生—人体解剖和组织胚胎学] R329.25[医药卫生—基础医学]