研究特异性抗HPV16E6核酶对宫颈癌细胞凋亡的影响。方法：设计特异性切割HPV16E6基因的核酶，构建抗HPV16E6核酶的真核表达质粒。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,CaSKi-P细胞。Northern杂交细胞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，并测定HPV16E6，c-myc，bal-2,p53,fas等蛋白的表达。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-mediated dUTP nick end labelling）2种方法测定细胞凋亡。结果：Northern杂交证实CaSKi-P中表达E6较CaSKi-P，CaS-Ki明显降低。与CaSKi-R细胞表达HPV16E6，c-myc,bcl-2蛋白明显减少，而表达p53明显增高；两者中fas蛋白的表达相近。CaSKi-P细胞中各基因的表达与CaSKi细胞无显著差异。结论：抗HPV16E6-Ribozyme的导入能诱导宫颈癌细胞凋亡，其原因可能在于病毒癌基因E6表达的降低，以及由此而引起的细胞内一系列基因表达的改变。

Objective: To study the characterization of the cultured cervical cancer cell line transfected with anti- HPV16E6-ribozyme, and to investigate the effect of ribozyme on proliferation and apoptosis of cervical cancer cell. Metliods: Anti-HPV16E6-ribozyme had been designed to cleave the HPV16E6 gene. With the method of lipofectin transfec- tion, the anti-HPVI6E6-ribozyme and empty eucaryotic expressing plasmids were transfected into CaSKi cell, which named as CaSKi-R, CaSKi-P respectively. The amounts of E6 mRNA in the three kinds of cells were detected by northern blot. Cell cycle was detemined by flow cytometry, and cell apoptosis was examined by fluorescent (Hoechst) staining and TUNEL. The expression of some genes, including c-myc, bcl-2, p53, and fas, was also detected by flow cytometry analy- sis. Results: Northern blot showed that E6 mRNA was less in CaSKi-R than in CaSKi. In CaSKi-R cells, cycle was arres- ted in G1 phase, with decreasing in percentage of S phase cells. The apoptosis rate of CaSK1-R cell was much higher than those of CaSKi and CaSK1-P. Anti-HPV16E6-ribozyme could reduce the expression of E6, c-myc, bcl-2 genes on CaSKi- R cells, and increased the expression of p53. While this phenomenon was not found on the CaSK1-P cells. The expression of fas was similar in the three kinds of cells. Conclusion: Anti-HPVE6-rivozyme induces apoptosis of human cervical cancer CaSKi cells. The mechanisms may be the decrease of E6 gene's expression, and the succedent changing of some genes'expression.

广东省自然科学基金资助项目(96058)