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目的:探讨纺锤体检查点蛋白E(Cenp-E)基因在肝癌细胞中的定位和作用,为进一步阐明肝癌细胞染色体数目异常的机理奠定基础。方法:利用FQ-PCR检测Cenp-E基因在用Nocodazole(诺考达唑)同时处理HepG-2细胞和LO2细胞中mRNA的表达水平。用间接免疫荧光技术观察Cenp-E蛋白在用Nocodazole(诺考达唑)同时处理HepG-2细胞和LO2细胞中的定位及表达。在LO2细胞中用RNAi技术干扰Cenp-E基因后,用FQ-PCR检测Cenp-E基因在干扰前后mRNA表达水平。并利用间接免疫荧光进一步评价干扰后的细胞功能情况。结果:Nocodazole处理前Cenp-E基因和蛋白在LO2和HepG-2细胞中表达无显著差异,处理后Cenp-E基因和蛋白在两种细胞表达都增高,但LO2细胞上调水平大于HepG-2细胞,差异具有统计学意义。间接免疫荧光结果显示Cenp-E蛋白主要定位细胞核内,并且在出现异常分裂的细胞核内Cenp-E蛋白的表达明显低于正常分裂的细胞核。在干扰Cenp-E后,间接免疫荧光显示干扰后的LO2细胞中出现核异常比例明显高于正常细胞。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 investigate the localization and function of Cenp-E gene in hepatocellular carcinoma cells and lay the foundation for further elucidating the mechanism of chromosomal abnormalities in hepatocellular carcinoma cells. Methods: The expression of Cenp-E gene in HepG-2 cells and LO2 cells treated with Nocodazole was detected by FQ-PCR. Indirect immunofluorescence was used to observe the localization and expression of Cenp-E protein in HepG-2 cells and LO2 cells treated with Nocodazole simultaneously. After interfering with Cenp-E gene by RNAi in LO2 cells, the level of mRNA expression of Cenp-E before and after interference was detected by FQ-PCR. Indirect immunofluorescence was used to further evaluate the cellular function after interference. Results: Cenp-E gene and protein were not significantly different between LO2 and HepG-2 cells before treatment with Nocodazole. The expression of Cenp-E gene and protein in both cells increased after treatment, but the level of LO2 cells was higher than that of HepG-2 cells , The difference was statistically significant. Indirect immunofluorescence results showed that Cenp-E protein mainly located in the nucleus, and the expression of Cenp-E protein was significantly lower than that of the normal dividing nucleus in the abnormally dividing nucleus. After interfering with Cenp-E, indirect immunofluorescence showed that the proportion of nuclear abnormalities in LO2 cells after interference was significantly higher than that in normal cells. Conclusion: Down-expression of Cenp-E may be one of the important reasons for the abnormal chromosome number of HCC cells.