Cenp-E表达下调与HepG-2细胞染色体数目异常的关系研究

摘要

目的 在人肝癌细胞(HepG-2细胞)中研究纺锤体检查点蛋白E(Cenp-E)的定位和作用.方法 用流式细胞术检测经诺考达唑处理后HepG-2细胞和正常肝细胞(LO2细胞)周期的变化,用染色体分析方法检测两种细胞染色体数量及核型,用实时荧光定量-聚合酶链反应(RFQ-PCR)检测经诺考达唑处理后HepG-2细胞和 LO2 细胞中Cenp-E mRNA 的表达水平,用间接免疫荧光技术观察两种细胞Cenp-E蛋白表达及定位,在LO2细胞中用 RNA干扰(RNAi)技术进一步评价Cenp-E蛋白的功能.结果 诺考达唑处理后,使两种细胞有丝分裂均停留在G2/M期;染色体分析结果显示HepG-2 细胞中染色体数目异常细胞的比例高于 LO2 细胞(P<0.05);RFQ-PCR显示诺考达唑处理后LO2细胞Cenp-E mRNA高于HepG-2 细胞(P<0.05);间接免疫荧光技术显示Cenp-E定位于细胞核,诺考达唑处理后LO2细胞Cenp-E蛋白表达高于HepG-2细胞(P<0.05);转染质粒后,LO2细胞中Cenp-E mRNA和蛋白的表达明显降低(P<0.05).结论 Cenp-E 过低表达可能是肝癌细胞染色体数目异常重要原因之一.%Objective To study the role and localization of Cenp-E gene in HepG-2 cell. Methods The cell cycle changes of HepG-2 cell and LO2 cell synchronized by nocodazole were detected by flow cytometry. The chromosome assay was uesd to detected the number and karyotype of chromosome of HepG-2 cell and LO2 cell. Cenp-E mRNA expressions in HepG-2 cell and LO2 cell synchronized by nocodazole were detected by RFQ-PCR. In the mean while. Cenp-E protein localization and expressions in HepG-2 cell and LO2 cell were oberved by indirect immunofluorescence. In the end,RNAi technology was used to further evaluated the function of Cenp-E in LO2 cell. Results HepG-2 cell and LO2 cell were arrested in G2/M phase synchronized by nocodazole. The chromosome assay showed that the proportion of abnormal chromosomes cell in HepG-2 cell were higher then LO2 cell. After synchronized by nocodazole,Cenp-E mRNA and protein expressions of HepG-2 cell was higher then LO2 cell. Cenp-E mRNA and protein expressions of LO2 cell decreased significantly. Conclusion The low expression of Cenp-E may be one of the important reasons of chromosome numerical abnormality in human hepatoma cells.