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背景与目的:人乳头瘤病毒与宫颈癌的发生相关,核酶是一种能切割靶RNA的特殊RNA。本研究旨在探讨转染了靶向切割HPV16E6mRNA核酶的宫颈癌细胞株的表型,以及核酶对宫颈癌细胞增殖与调亡的作用。方法:计算机设计特异性切割HPV16E6mRNA的核酶,构建抗HPV16E6核酶(HRz)的真核表达质粒。以脂质体法将抗HPV16E6核酶、空载体质粒分别导入CaSKi细胞,命名为CaSKi-R和CaSKi-P细胞。Northern杂交检测3种细胞中E6基因的表达。流式细胞仪检测3种细胞的凋亡率,将3种细胞在相差显微镜和荧光显微镜下观察,采用荧光(Hoechst33342)染色和TUNEL(TDT-mediateddUTPnickendlabeling)两种方法测定细胞凋亡。流式细胞术测定HPV16E6、c-myc、bcl-2、p53、Fas等蛋白的表达。结果:RNA点杂交证实核酶mRNA能稳定表达于CaSKi-R细胞中。Northern杂交证实CaSKi-R中E6mRNA表达水平较CaSKi细胞明显降低,而CaSKi-P和CaSKi细胞的E6mRNA表达水平无差异。CaSKi-R细胞凋亡率明显高于CaSKi和CaSKi-P细胞,细胞周期阻滞于G2期,S期细胞百分率下降。抗HPV16E6核酶能明显减少CaSKi-R细胞中HPV16E6、c-myc、bcl-2蛋白的表达,增高p53蛋白的表达;这种现象并不发生于CaSKi-P细胞中。Fas蛋白的表达在3种细胞中都相近。结论:抗HPV16E6核酶的导入能诱导宫颈癌细
BACKGROUND & OBJECTIVE: Human papillomavirus (HPV) is associated with the development of cervical cancer. Ribozyme is a special RNA that cleaves target RNA. The purpose of this study was to investigate the phenotypes of cervical cancer cell lines transfected with HPV16E6mRNA targeting ribozymes and the effect of ribozymes on the proliferation and apoptosis of cervical cancer cells. Methods: The HPV16E6mRNA ribozyme was specifically designed by computer to construct eukaryotic expression plasmid of anti-HPV16E6 ribozyme (HRz). The anti-HPV16E6 ribozyme and empty vector plasmid were respectively introduced into CaSKi cells by liposome method and named as CaSKi-R and CaSKi-P cells. Northern blot analysis of E6 gene expression in three kinds of cells. The apoptosis rates of the three kinds of cells were detected by flow cytometry. The three kinds of cells were observed under the phase contrast microscope and the fluorescence microscope. The apoptosis was determined by fluorescence (Hoechst33342) staining and TUNEL (TDT-mediated dUTPnickendlabeling). Flow cytometry was used to detect the expression of HPV16E6, c-myc, bcl-2, p53, Fas and other proteins. Results: RNA dot blot confirmed that ribozyme mRNA was stably expressed in CaSKi-R cells. Northern blot showed that the expression level of E6 mRNA in CaSKi-R was significantly lower than that in CaSKi cells, while there was no difference in E6 mRNA expression between CaSKi-P and CaSKi cells. The apoptosis rate of CaSKi-R cells was significantly higher than that of CaSKi and CaSKi-P cells, the cell cycle arrest in G2 and S phase cells decreased. Anti-HPV16E6 ribozyme can significantly reduce the expression of HPV16E6, c-myc, bcl-2 protein and increase the expression of p53 protein in CaSKi-R cells; this phenomenon does not occur in CaSKi-P cells. The expression of Fas protein was similar in all three kinds of cells. Conclusion: The introduction of anti-HPV16E6 ribozyme can induce cervical cancer