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[目的]研究转染人乳头状瘤病毒18型(HPV18)后,甲基硝基亚硝基胍(MNNG)暴露对人食管上皮Het-1A细胞周期和增殖、凋亡功能的影响。[方法]通过慢病毒介导稳定转染HPV18 E6、E7基因到人食管上皮Het-1A细胞中,荧光定量PCR检测E6和E7相对表达量后得到稳定表达株。应用MTT法观察MNNG(0、1、5、10μmol/L)染毒处理24 h后对转染组和对照组细胞增殖功能的影响。应用流式细胞术检测转染组和对照组细胞染毒后细胞周期和凋亡的变化。[结果]HPV18转染组与对照组相比,细胞增殖能力增强,G1期延长,S期缩短(P<0.05),且凋亡率下降(P<0.05)。非转染细胞中MNNG染毒各组与未染毒组相比,细胞增殖活性受到抑制,G1期延长,细胞凋亡率增加(P<0.05)。HPV18转染后MNNG暴露,可导致G2期延长;MTT检测细胞增殖结果显示HPV18转染组细胞在MNNG作用下,细胞存活率均高于同浓度MNNG单独暴露的非转染组细胞;中、低浓度MNNG暴露后HPV18转染组较未转染组相比细胞凋亡率降低(P<0.05)。[结论]Het-1A细胞转染HPV18后暴露MNNG,细胞凋亡受到一定程度抑制,促进细胞增殖。由此可能导致细胞不断积累MNNG引起的基因突变和DNA损伤。
[Objective] The purpose of this study was to investigate the effects of methyl nitroso-nitrosoguanidine (MNNG) exposure on the cell cycle, proliferation and apoptosis of human esophageal epithelial Het-1A cells after transfection with human papillomavirus type 18 (HPV18). [Methods] The HPV18 E6 and E7 genes were transfected into human esophageal epithelial Het-1A cells by lentivirus-mediated transfection. The relative expression levels of E6 and E7 were detected by fluorescence quantitative PCR and the stable expression strains were obtained. The effect of MNNG (0, 1, 5, 10μmol / L) on the cell proliferation was observed by MTT assay 24 h after transfection. Flow cytometry was used to detect the changes of cell cycle and apoptosis after transfection and control cells were infected. [Results] Compared with the control group, HPV18 transfected cells enhanced the cell proliferation, prolonged the G1 phase, shortened the S phase (P <0.05) and decreased the apoptosis rate (P <0.05). Compared with non-transfected MNNG group, the cell proliferation activity was inhibited, the G1 phase prolonged and the apoptosis rate increased (P <0.05). The results of MTT assay showed that the cell viability of MN18 cells transfected with HPV18 was higher than that of untransfected cells exposed with MNNG alone, Compared with untransfected group, the apoptosis rate of HPV18 transfected group after MNNG exposure was lower (P <0.05). [Conclusion] MNNG was exposed in Het-1A cells transfected with HPV18, apoptosis was inhibited to a certain degree and cell proliferation was promoted. This may lead to cell accumulation of MNNG-induced mutations and DNA damage.