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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 a CENP-I specific RNA interference eukaryotic expression vector and observe the effect of CENP-I gene expression on the growth, cell cycle and chromosomal abnormalities in HEK293 cells in vitro. Methods CENP-IsiRNA eukaryotic expression vector was constructed and transfected into pGenesil-1 empty vector and pGenesil-1 / CENP-I-siRNA-1, pGenesil-1 / CENP- / CENP-I-siRNA-3 eukaryotic expression vector were introduced into human embryonic kidney HEK293 cells. The cells were collected 24, 48 and 72 hours after transfection. The expression of CENP-I protein and mRNA in HEK293 cells were detected by Western blot and FQ-PCR to determine the interference effect and the best interference time. The cell growth of each experimental group was detected by MTT method. The cell cycle distribution of each group was detected by flow cytometry. The cell division index was counted by Giemsa staining method. Chromosome specimens were prepared by routine method. Results The CENP-I siRNA eukaryotic expression vector was successfully constructed and the plasmid vector pGenesil-1 / CENP-I-siRNA-3 with interference effect was selected. After transfection with HEK293 cells for 72 hours, CENP-I protein and mRNA Significantly expressed under the withered. The growth of HEK293 cells with decreased expression of CENP-I was significantly slowed down (P <0.05), the cells increased at G2 / M phase, and the percentage of cells at dividing stage increased. Conclusion RNA interference effectively and specifically inhibits the centromere-specific protein CENP-I expression. Inhibition of CENP-I can slow down the growth of human embryonic kidney HEK293 cells, decreased proliferation, cell division to extend.