人乳头瘤病毒18型E6的点突变体对宫颈癌Hela细胞生长和凋亡的影响  

Mutation of HPV 18 E6 inhibits the growth of Hela cells and induces apoptosis

作　　者：左丽君[1] 吴宝杰[1] 刘飞[1] 吴燕雯[1] 范立强[1] 

机构地区：[1]生物反应器工程国家重点实验室华东理工大学,上海200237

出　　处：《中国生化药物杂志》2014年第1期51-55,共5页Chinese Journal of Biochemical Pharmaceutics

摘　　要：目的构建并鉴定pcDNA 3.1(+)/HPV 18 E6及其突变体pcDNA 3.1(+)/E6 F49R、pcDNA 3.1(+)/E6 F1 27R真核重组表达质粒,并在转染Hela细胞后,检测其对宫颈癌Hela细胞生长增殖的影响。方法用RT-PCR法从HeLa细胞中扩增HPV 18 E6基因及其突变体HPV 18 E6 F49R、HPV 18 E6 F1 27R、HPV 1 8 E6 F49R-F127R基因片段,构建重组表达载体pcDNA 3.1(+)/HPV 1 8 E 6、pcDNA 3.1(+)/E6 F49R、pcDNA 3.1(+)/E6 F1 27R,在HeLa细胞中进行转染,48 h后进行MTT试验,利用吖啶橙/溴乙锭(AO/EB)双染法在荧光显微镜下进行细胞形态学的观察。结果成功构建了3种重组表达载体;重组质粒成功转染到Hela细胞中,pcDNA 3.1(+)/HPV 18 E6能促进细胞增殖;pcDNA 3.1(+)/E6 F 49 R和pcDNA 3.1(+)/E6 F127R在一定程度上可抑制表达有E6全长的HPV阳性细胞系Hela的增殖;荧光显微镜下观察到转染了突变体重组质粒的细胞均呈现细胞核皱缩,表现为早起凋亡现象。结论HPV 18 E6及其突变体的真核表达为研究其表达作用机制及为研究预防和治疗子宫癌奠定基础。Objective To construct pcDNA 3.1(+)/HPV 18 E 6 fusion gene and a single-codon mutation pcDNA 3.1(+)/E6 F49R or pcDNA 3.1(+)/E6 F127R fusion gene in eukaryotic expression vector and study the effects on proliferation and apoptosis of cervical carcinoma cell line Hela. Method HPV 18 E6 gene sequence and the single-point mutation HPV 18 E6 F49R or HPV 18 E6 F127R were amplified from total RNA of Hela cell line by reverse transcription- polymerase chain reaction ( RT- PCR), then the gene sequences were respectively inserted into pcDNA 3.1(+) vector to reconstruct recombinant plasmids which were transfected transiently into Hela cells. MTT and RT-PCR were used to test the expression levels of HPV 18 E6 and the growth of HeLa cells after transfected about 48 h. The proliferation and apoptosis of Hela cells were detected respectively by cell counting and AO/EB fluorescent vital staining. Results The pcDNA 3.1(+)/HPV 18 E6, pcDNA 3.1(+)/E6 F49R and pcDNA 3.1(+)/E6 F127R eukaryotic expression vectors were successfully constructed. The gene of HPV 18 E6 was discriminably detected in the HeLa cells which were transfected with the recombinant plasmids. After several days, the proliferation of Hela cells transfected with pcDNA 3.1(+)/E6 F49R or pcDNA 3.1(+)/E6 F127R plasmid were obviously inhibited and the apoptotic rates were significantly increased, then the proliferation of cells transfected with pcDNA(+)/HPV 18 E6 was rather increased slightly, and we could observe the phenomena of early apoptosis and the formation of thekaryopyknosis by fluorescent microscope in the cells transfected with pcDNA 3.1(+)/E6 F49R or pcDNA 3.1(+)/E6 F127R. Conclusion The eukaryotic expressing vectors encoding HPV 18 E6 F49R and HPV 18 E6 F127R provide fundamental basis for the further study on HPV 18 E6 mechanism as well as prevention and treatment of uterine cancer.

关 键 词：人乳头瘤病毒HPV 真核表达 HELA细胞 

分 类 号：Q78[生物学—分子生物学]