人工合成的亚硝基化合物MNNG致人胃黏膜上皮细胞恶性转化模型的建立及机制分析  

Establishment of MNNG-induced malignant transformation model of human gastric mucosal epithelial cells

作　　者：王霞 叶景鸿 许尤琪 WANG Xia;YE Jinghong;XU Youqi(The Second Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210017,China)

机构地区：[1]南京中医药大学第二附属医院(江苏省第二中医院),南京210017

出　　处：《山东医药》2021年第2期46-49,共4页Shandong Medical Journal

基　　金：江苏省自然科学青年基金资助项目(BK20171096);全国中医药创新骨干人才培训项目(国中医药人教函2019-128号)。

摘　　要：目的建立人工合成的亚硝基化合物N-甲基-N-硝基-N-亚硝基胍(MNNG)暴露致人胃黏膜上皮细胞GES-1恶性转化的模型,并分析其作用机制。方法取GES-1细胞,分为空白对照组、溶剂对照组和MNNG诱导组。空白对照组正常培养,MNNG诱导组在培养基中加入2μmol/L的MNNG,溶剂对照组加入等体积的DMSO。常规培养4周。于实验第5周行病理染色观察三组细胞形态,细胞克隆形成实验检测细胞克隆能力,划痕实验检测细胞迁移能力,Western blotting法检测细胞自噬相关蛋白LC3Ⅱ、Beclin1表达,qPCR法检测LC3Ⅱ、Beclin1 mRNA表达,裸鼠成瘤实验检测各组细胞成瘤能力。结果MNNG诱导组细胞体积增大,形态不规则,排列紊乱,部分细胞呈团块状生长,部分细胞呈梭形。与空白对照组相比,MNNG诱导组细胞克隆形成数目明显增多,细胞迁移速度增高(P均<0.05),LC3Ⅱ、Beclin1蛋白及mRNA表达水平降低(P均<0.05),裸鼠成瘤能力增加。结论成功建立MNNG致人胃黏膜上皮细胞恶性转化模型,其机制可能与自噬相关。Objective To establish a model of malignant transformation of human gastric mucosal epithelial cells GES-1 by N-methyl-N-nitro-N-nitrosoguanidine(MNNG)exposure and to explore the mechanism.Methods GES-1 cells were divided into three groups:the blank control group,solvent control group,and MNNG-induced group.The cells in the blank control group were cultured normally.The cells in the MNNG-induced group were added with 2μmol/L MNNG in the medium,and the solvent control group with equal volume of DMSO.GES-1 cells were treated for 4 consecutive weeks.On the 5th week,the cell morphological changes in each group were observed.Cell clone formation experiment was performed to test the cell cloning ability,Scratch test to detect cell migration ability,and Western blotting to detect the expression levels of autophagy-related proteins LC3Ⅱand Beclin1,qPCR to detect the expression levels of LC3Ⅱand Beclin1 mRNA,and nude mice tumor formation test to detect the tumor formation ability of cells in each group.Re⁃sults In the MNNG-induced group,the volume of cells increased,the morphology was irregular,and the arrangement was disordered;some cells grew into clumps,and some cells became spindle-shaped.Compared with the blank control,the number of cell clone formation in the MNNG-induced group increased significantly,cell migration capacity was significantly enhanced,the protein and mRNA expression levels of LC3Ⅱand Beclin1 decreased(all P<0.05),and the tumor formation ability in the nude mice increased in the MNNG-induced group.Conclusion The model of MNNG-induced malignant transformation of human gastric mucosal epithelial cells is established successfully and it might be related to autophagy.

关 键 词：人胃黏膜上皮细胞 亚硝基化合物 N-甲基-N-硝基-N-亚硝基胍 细胞恶性转化 细胞自噬 细胞模型 

分 类 号：R735.2[医药卫生—肿瘤]