MNNG和佛波酯诱导建立结肠癌的恶性转化细胞模型  

Establishment of malignant transformation cell model of colon cancer induced by MNNG and phorbol 12-myristate 13-acetate

作　　者：魏刚 袁利娟 路建国 刘沣涛 段森森 包国强 WEI Gang;YUAN Lijuan;LU Jianguo;LIU Fengtao;DUAN Sensen;BAO Guoqiang(Shaanxi University of Chinese Medicine,Xianyang 712046,Shaanxi,China;Department of General Surgery,the Second Affiliated Hospital of AiRForce Medical University,Xi′an 710038,Shaanxi,China)

机构地区：[1]陕西中医药大学,陕西省咸阳市712046 [2]空军军医大学第二附属医院普通外科,陕西省西安市710038

出　　处：《广西医学》2023年第16期1987-1991,共5页Guangxi Medical Journal

摘　　要：目的建立结肠癌的恶性转化细胞模型。方法以1-甲基-3-硝基-1-亚硝基胍(MNNG)为启动剂、佛波酯(PMA)为促癌剂,对永生化的正常人结肠上皮细胞(NCM460细胞)进行多代诱导处理。采用MTT实验、细胞克隆形成实验和细胞划痕实验鉴定NCM460细胞恶性转化的倾向性和恶性生物学特征,采用Western blot检测神经钙黏附蛋白(N-cadherin)、转化生长因子β1(TGF-β1)和表皮生长因子受体(EGFR)的蛋白表达水平。结果正常NCM460细胞形态为梭形,排列有序,呈单层生长;诱导后的第30代NCM460细胞形态发生显著变化,大小不一,排列紊乱,呈团块状生长。正常NCM460细胞、第17代NCM460细胞、第30代NCM460细胞的增殖能力依次升高,克隆形成数量依次增多,48 h迁移距离和迁移率依次增加或升高(P<0.05)。第30代NCM460细胞中N-cadherin、EGFR、TGF-β1的蛋白表达水平高于正常NCM460细胞(P<0.05)。结论经过MNNG和PMA连续多代诱导处理后,NCM460细胞的生物学功能(增殖、克隆形成和迁移能力)发生明显改变,具备恶性细胞的生物学特征。该模型可用于结肠癌的发病机制和治疗方法研究。Objective To establish the malignant transformnation cell model of colon cancer.Methods Immortalized normal human colonic epithelial cells(NCM460 cells)were induced for multiple generations with N-methyl-n'-nitro-N-nitrosoguanidine(MNNG)as the initiator and phorbol 12-myristate 13-acetate(PMA)as the tumor promoter.The MTT assay,cell colony formation assay,and cell scratch assay were used to validate the malignant transformation tendency and malignant biological characteristics of NCM460 cells.The protein expressions of neural-cadherin(N-cadherin),transforming growth factor beta 1(TGF-β1)and epidermal growth factor receptor(EGFR)were detected by the Western blot.Results The morphology of normal NCM460 cells was spindle-shaped,arranged in order,and grew in a monolayer.After induction,the morphology of the 30^(th) generation of NCM460 cells changed significantly,with different sizes,disordered arrangement,and clumped growth.The proliferation ability of normal NCM460 cells,the 17^(th) generation of NCM460 cells and the 30^(th) generation of NCM460 cells increased successively,the number of colony formation increased successively,and the migration distance and migration rate at 48 hours were increased or elevated successively(P<0.05).The protein expressions of N-cadherin,EGFR and TGF-β1 in the 30^(th) generation of NCM460 cells were higher than those in normal NCM460 cells(P<0.05).Conclusion After continuous multiple generations of induction with MNNG and PMA,the biological functions(proliferation,colony formation,and migration)of NCM460 cells are significantly changed,and NCM460 cells have the characteristics of malignant cells.This model can be used to study the pathogenesis and therapeutic regimen of colon cancer.

关 键 词：结肠癌 1-甲基-3-硝基-1-亚硝基胍 佛波酯 人结肠上皮细胞 恶性转化细胞模型 

分 类 号：R735.35[医药卫生—肿瘤] R3[医药卫生—临床医学]