摘要
目的构建人乳头瘤病毒(HPV)18型 E6/E7反义荧光真核表达载体,研究其对宫颈癌 HeLa 细胞增殖凋亡的影响。方法以 PCR 法扩增 HPV18型 E6/E7区716bp 片段并反向克隆到荧光真核表达载体 pEGFP-C1,构建重组体 pEGFP-HPV18E6E7as(EGFP-18AS)并转染人宫颈癌 HeLa细胞。利用逆转录聚合酶链反应(RT-PCR)和 Western 印迹技术检测转染后 E6、E7基因在 mRNA 和蛋白水平的变化;用四甲基偶氮唑盐微量酶反应比色法检测转染后细胞的的增殖活性;用流式细胞仪检测转染后细胞的凋亡率;荧光显微镜下观察凋亡细胞的形态学改变。结果转染 EGFP-18AS 重组体后,HeLa 细胞中 E6、E7基因的 mRNA 表达和蛋白合成均明显下调;细胞增殖受到抑制;流式显示细胞 G_1期明显阻滞;同时在荧光显微镜下经 Hoechst 染色凋亡细胞出现染色质凝集、边集、碎裂等形态学改变。结论重组质粒 pEGFP-HPV18E6E7as 能有效地抑制人宫颈癌 HeLa 细胞的生长和增殖,诱导其凋亡,证明了反义 RNA 技术的有效性,为宫颈癌的基因治疗提供了一种新的可能的途径。
Objective To investigate the effect of eukaryotic fluorescent expression vector carrying antisense human papillomavirus （HPV） 18 E6/E7 on the growth and proliferation of human cervical carcinoma. Methods The HPV18 E6E7 with the length of 716 bp was amplified by PCR, the PCR product was inversely inserted into the eukaryotic fluorescent expression vector pEGFP-C1 so as to construct the recombinant eukaryotic expression plasmid pEGFP-HPV18E6E7as （EGFP-18AS） . Human cervical carcinoma cells of the line HeLa were cultured and randomly divided into 3 groups： Group A transfected with the recombinant plasmid EGFP-18AS, Group B transfected with the blank plasmid pEGFP-C1, and Group C without transfection used as control group. The mRNA expression of HPV 18 E6/E7 in the HeLa cells was detected by RT-PCR and protein expression of HPV18 E6/E7 HPV 18 E6/E7 in the HeLa cells was detected by. Western blotting MTT assay was performed to dynamically monitor the surviving cells and the cell apoptosis was observed by flow cytometry and fluorescence microscopy. Results The protein and mRNA expression levels of HPV18 E6/E7 in the HeLa cells transfected with HeLa/18AS were both remarkably lower than those in the HeLa cells transfected with blank plasmid and those of the control group. The numbers of surviving HeLa cells of Group A was significantly lower than those of Groups B and C （ both P 〈 0. 05）. The phenomenon of arrest of Gt phase was remarkable in Group A. The apoptotic rate of the cells of Group A was 47.21%, significantly higher than those of Groups B and C （14. 18% and 3. 36% respectively, both P 〈 0. 05 ） . An increased number of cells with chromosome condensation and fragmentation was found in Group A as compared with Groups B and C. Conclusion The recombinant pEGFP-HPV18E6ETas can effectively inhibit the growth and proliferation of human cervical carcinoma HeLa cells, and further induce the cell apoptosis. The antisense RNA technology is available and may provide a new way to gene therapy of the cervical carcinoma.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2007年第23期1618-1621,共4页
National Medical Journal of China
基金
国家自然科学基金(30528012
30672227)
国家重点基础研究发展计划(973)基金(2002CB513100)
关键词
宫颈肿瘤
乳头瘤病毒
人
凋亡
Uterine Neoplasms
Papillomavirus, Human
Apoptosis
