癌基因高表达人上皮细胞转化模型的构建及应用  

Establishment and application of oncogene over expressed human epithelial cell transformation model

作　　者：马儒林[1] 庞雅琴[1] 李文学[1] 肖勇梅[1] 魏青[1] 李道传[1] 赖延东[1] 林育纯[1] 王庆[1] 杨萍[1] 陈丽萍[1] 唐石伏[1] 林忠宁[1] 陈雯[1] 

机构地区：[1]中山大学公共卫生学院预防医学研究所,广州510080

出　　处：《中华预防医学杂志》2008年第6期395-399,共5页Chinese Journal of Preventive Medicine

基　　金：国家自然科学基金（30630055,30571588）;国家“973”计划（2002CB512903）;广东省自然科学基金（5001763）;新世纪优秀人才支持计划（05-0722）;高等学校博士学科点专项（20060558024）

摘　　要：目的构建癌基因高表达人支气管上皮细胞模型并应用于致癌物诱导细胞恶性转化活性的检测。方法在永生化人支气管上皮细胞株16HBE中依次导入人端粒酶催化亚基（hTERT）、癌基因H—Ras^V12。或c—Myc或空白载体，构建细胞株16HBETR、16HBETM和16HBETV。检测所构建细胞株的生物学特性如细胞形态、细胞生长、染色体畸变等指标。用已知致癌物硫酸镍（NiSO4）和二氢二醇环氧苯并（a）芘（BPDE）多次染毒，利用软琼脂试验及荷瘤试验检测化学物诱导细胞转化的活性。结果经过端粒酶活性测定以及蛋白印迹验证上述转基因16HBE细胞株均构建成功。癌基因H—Ras和c—Myc的表达分别使细胞的增殖加速30．3％和10．4％，但是细胞染色体的畸变率没有发生改变。癌基因高表达细胞株只在软琼脂中形成少数背景克隆，但不能在裸鼠皮下形成肿瘤。BPDE诱导16HBETR、16HBETM细胞发生恶性转化的间期分别为5周、11周，比对照组细胞16HBETV的加周分别提前了15周和9周；而硫酸镍诱导16HBETR、16HBETM细胞转化的间期分别为11周、14周，比对照组细胞的32周分别提前了21周和18周。结论癌基因高表达的细胞转化模型可大大缩短试验间期，有望应用于环境致癌物的筛查及化学致癌机制的研究。Objective To establish human bronchial epithelial cell lines over expressing oncogene and to investigate its application in detection of carcinogen-induced cell transformation. Methods Mediated by retrovirus infection, human telomerase catalytic subunit, hTERT was introduced into immortal human bronchial epithelial cells （16HBE） and followed by introduction of the oncogenic allele H-Ras（V12） , or cMyc or empty vector, creating cell lines 16HBETR, 16HBETM and 16HBETV, respectively. Biological characteristics of these cell lines including morphology, proliferation, and chromosomal aberration were examined to access whether they were transformed. Soft agar experiment and nude mice subcutaneous injection were performed using pre-transformed 16HBE cells induced by known carcinogens, nickel sulfate （ NiSO4 ） and 7, 8,-dihydrodiol-9, 10-epoxide benzo[ a ] pyrene （ BPDE ）. Results With detection of telomerase activity and Western blotting, the expression of target proteins was verifed. Thus, the transgenic 16HBE cell lines were successfully established. Cells expressing oncogene H-Ras or c-Myc grew 30.3% or 10.4% faster than control cells. However, these cells failed to form colonies in soft agar or form tumor in nude mice. 16HBETR, 16HBETM cells obtained transformed phenotype at 5 wks, 11 wks, respectively after treatment with BPDE, which are 15 wks and 9 wks earlier than control cells 16HBETV （20 wks）. Meanwhile, 16HBETR, 16HBETM cells obtained transformed phenotype at 11 wks, 14 wks, respectively after treatment with nickel sulfate, which are 21 wks and 18 wks earlier than control cells （32 wks）. Conclusion With the advantage of shorter latency, transgenic human cell transformation models could be used in potent carcinogen screening and applied to chemical-carcinogensis mechanism study.

关 键 词：细胞转化 肿瘤 上皮细胞 基因 ras 基因 myc 

分 类 号：R686[医药卫生—骨科学]