特异性核酶对宫颈癌细胞系CaSKi增殖与凋亡的影响  

Effects of anti-HPV16E6-ribozyme on the proliferation and apoptosis of human cervical cancer cell line CaSKi

作　　者：郑燕芳[1] 饶智国[1] 张积仁[1] 

机构地区：[1]第一军医大学珠江医院肿瘤中心,广东广州510282

出　　处：《第一军医大学学报》2002年第6期496-498,502,共4页Journal of First Military Medical University

基　　金：广东省自然科学基金(96058)

摘　　要：目的研究特异性抗HPV16E6核酶对宫颈癌细胞增殖与凋亡的影响。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。点杂交检测核酶在细胞中的表达,Northern blotting检测CaSKi、CaSKi-R、CaSKi-P 3种细胞中E6基因的表达。测定3种细胞的生长曲线和软琼脂克隆形成率,并以皮下接种法检测细胞在裸鼠体内的成瘤能力。流式细胞仪检测3种细胞的凋亡率,并测定c-myc、bcl-2、p53、fas、PCNA、C-erbB-2等基因的表达。结果点杂交证实该核酶能在CaSKi-R细胞中稳定表达,Northern blotting证实CaSKi-R中表达E6较CaSKi-P、CaSKi明显降低。与CaSKi细胞相比,CaSKi-R细胞生长速度、软琼脂克隆形成率明显降低,在裸鼠体内的致癌能力下降,凋亡率明显增高,出现凋亡峰,S期、G2M期细胞百分率下降;而CaSKi-P细胞无此改变。CaSKi-R细胞表达HPV16E6、C-myc、Bcl-2、PCNAC-erbB-2蛋白明显减少,而p53表达明显增高;CaSKi和CaSKi-R细胞中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核酶的导入能阻碍宫颈癌细胞增殖,诱导其凋亡,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?Objective To study the characterization of anti-HPV16E6-ribozyme transfected into cultured human cervical cancer cell line, and to investigate the effect of ribozyme on the proliferation and apoptosis of the cells. Methods By way of lipofectin transfection, anti-HPV16E6-ribozyme, a ribozyme designed to specifically cleave the HPV16E6 gene, and empty vector expression plasmids were respectively transfected into CaSKi cells which were subsequently designated as CaSKi-R and CaSKi-P accordingly. The expression of ribozyme in the transfected cells was observed by RNA dot blot analysis, and E6 mRNA expression was detected by Northern blotting in the 3 kinds of cells whose growth curves and clone-forming ability on soft agar were studied, with their tumorgenicity observed by percutaneous inoculation of the cells in nude mice. Flow cytometry was employed to assess the apoptosis rates and the expression of the genes including c-myc, bcl-2, p53, and fas etc. Results Stable expression of anti-HPV16E6-ribozyme was observed in CaSKi-R cells that had less E6 mRNA expression than CaSKi had as shown by Northern blotting. When compared with those of CaSKi cells, the growth rate and clone-forming ability on soft agar of CaSKi-R were reduced, while those of CaSKi-P evinced no significant changes. The tumorgenicity of CaSKi-R in nude mice was also comparatively decreased, with markedly elevated apoptosis rate. Anti-HPV16E6-ribozyme reduced the expressions of E6, c-myc, bcl-2, PCNA and C-erbB2 genes but increased p53 expression in CaSKi-R cells, which, however, did not happen in CaSKi-P cells. The expression of Fas underwent no significant changes in response to the transfection. Conclusion Anti-HPVE6-ribozyme inhibits proliferation and induces apoptosis in CaSKi cells, possibly due to the decrease of E6 gene expression that triggers a series of changes in the gene expressions of the cells.

关 键 词：特异性核酶 宫颈癌细胞系 CASKI 影响 脱噬作用 细胞分裂 细胞增殖 细胞凋亡 

分 类 号：R737.33[医药卫生—肿瘤]