HPV联合MNNG对Het-1A细胞恶性转化的影响

Influence of HPV on MNNG-induced malignant transformation of Het-1A cells

目的:探讨人乳头状瘤病毒(HPV)全长基因转染与N-甲基-N′-硝基-N-亚硝基胍(MNNG)染毒联合作用对食管正常上皮Het-1A细胞恶性转化的影响,为食管癌的发病机制提供理论依据。方法:以Het-1A细胞为靶细胞,设4组,包括空载体转染对照组、转染HPV-18组(HPV)、MNNG染毒组(MNNG)、HPV与MNNG联合作用组(HPV+MNNG),其中MNNG染毒剂量为2μmol/L,每代染毒1次,每次24h;选取第10代、20代及35代细胞进行形态学观察和功能学试验。采用CCK-8法检测各代各组细胞增殖活力,流式细胞术检测各代细胞周期分布,用增殖指数表示细胞的增殖状况。并采用AnnexinV-APC/PI双染法检测细胞凋亡,通过侵袭迁移能力以及软琼脂集落形成试验检测细胞恶性转化表型,最后通过裸鼠成瘤试验观察恶性转化细胞在体内的增殖状况。结果:随着染毒代数增加,HPV+MNNG组与MNNG组细胞形态发生了明显的改变,连接松散、细长、伴伪足。CCK-8检测结果与细胞周期检测结果一致,与对照组比较,HPV+MNNG组和MNNG组的细胞增殖活性出现了先升高再下降最后升高的趋势(P均<0.05),且HPV+MNNG组细胞变化更明显。与MNNG单独作用组比较,HPV+MNNG联合作用组细胞的抗凋亡能力增强(P<0.05)。侵袭和迁移试验结果显示HPV+MNNG组穿膜细胞数均显著高于其他各组(P<0.05),MNNG组次之;软琼脂集落形成试验中,HPV+MNNG组细胞集落形成率[(30.8±0.2)%]高于MNNG组[(27.1±0.3)%](P<0.05)。HPV+MNNG组与MNNG组裸鼠成瘤率均为100%,瘤体大小无显著性差异。HPV单独作用组与对照组裸鼠未成瘤。结论:HPV与MNNG联合作用可致食管正常上皮细胞Het-1A恶性转化,使细胞增殖能力、抗凋亡能力、侵袭迁移能力及非锚定独立生长能力均显著增强,参与并促进恶性转化进程。

OBJECTIVE:To explore the influence of expression of human papilloma virus(HPV)on MNNGinduced malignant transformation of Het-1A normal esophageal epithelial cells,and to provide better understanding for pathogenesis of esophageal cancer.METHODS:Cells were divided into HPV(transferred with HPV lentivirus),control(transferred with empty vector),MNNG,and combined HPV and MNNG groups.HPV expression and empty carrier cells were exposed to MNNG(2 mol/L)for 24 hours per generation.The morphology of these cells was observed at the same th th th time.Functional tests were conducted in cells of the 10,20 and 35 generations.CCK-8 and cell cycle distribution detection assays were conducted to document changes of viability and proliferation activity.Proliferation indexes were used to monitor proliferation.Apoptosis was detected by Annexin V-APC/PI double staining.Invasion and migration ability of cells were detected by transwell assay.Soft agar colony forming experiments were performed.Nude mice tumorigenicity assay was conducted to detect malignantly transformation.RESULTS:Among the different cell groups,the MNNG and the combined HPV and MNNG groups had the most obvious change in morphology,with the formation of loose,slender and pseudopods.These observations were consistent with that from the CCK-8 and the cell cycle distribution assays.The proliferative activities for the MNNG and the combined HPV and MNNG groups were increased first,then inhibited and finally increased again(P<0.05).Compared with the MNNG exposure group,the anti-apoptotic ability of the combined HPV and MNNG group was increased(P<0.05).The results of invasion and migration tests showed that the number of transmembrane in cells of the combined HPV and MNNG group was significantly higher than that in the other groups(P<0.05)which was followed by the MNNG exposure group.In the soft agar cloning experiment,the colony formation rate of the combined HPV and MNNG group was(30.8±0.2)%higher than that of the MNNG group(27.1±0.3)%(P<0.05).Cells from the MNNG and the combined groups induced tumors in nude mice while the HPV and the control groups did not form any local mass.CONCLUSION:Combined exposure of HPV and MNNG caused malignant transformation of the Het-1A normal esophageal epithelial cells via significant enhancement of cell proliferation,anti-apoptosis,invasion,cell migration and non-anchorage independent growth.HPV expression could therefore participate in and promote the malignant transformation process.