CAJ | 학술논문

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 F127R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 F127R、HPV 18 E6 F49R-F127R基因片段，构建重组表达载体pcDNA 3.1(+)/HPV 18 E 6、pcDNA 3.1(+)/E6 F49R、pcDNA 3.1(+)/E6 F127R，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。
목적：구건병감정pcDNA 3.1(+)/HPV 18 E6급기돌변체pcDNA 3.1(+)/E6 F49R、pcDNA 3.1(+)/E6 F127R진핵중조표체질립，병재전염Hela세포후，검측기대궁경암Hela세포생장증식적영향。방법용RT-PCR법종HeLa세포중확증HPV 18 E6기인급기돌변체HPV 18 E6 F49R、HPV 18 E6 F127R、HPV 18 E6 F49R-F127R기인편단，구건중조표체재체pcDNA 3.1(+)/HPV 18 E 6、pcDNA 3.1(+)/E6 F49R、pcDNA 3.1(+)/E6 F127R，재HeLa세포중진행전염，48 h후진행MTT시험，이용아정등/추을정(AO/EB)쌍염법재형광현미경하진행세포형태학적관찰。결과성공구건료3충중조표체재체；중조질립성공전염도Hela세포중；pcDNA 3.1(+)/HPV 18 E6능촉진세포증식；pcDNA 3.1(+)/E6 F 49 R화pcDNA 3.1(+)/E6 F127R재일정정도상가억제표체유E6전장적HPV양성세포계Hela적증식；형광현미경하관찰도전염료돌변체중조질립적세포균정현세포핵추축，표현위조기조망현상。결론HPV 18 E6급기돌변체적진핵표체위연구기표체작용궤제급위연구예방화치료자궁암전정기출。
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.