MNNG诱导TERC基因沉默的16HBE细胞恶性转化模型的建立  

A malignant transformation model of TERC-deficient 16HBE cells induced by MNNG

作　　者：朱俊峰[1] 黄小荣[2] 冯宇鹏[3] 周俊宜[1] 

机构地区：[1]中山大学中山医学院生物化学与分子生物学教研室,广东广州510080 [2]中山大学中山医学院实验教学中心,广东广州510080 [3]广东医学院附属西乡人民医院泌尿外科,广东深圳518102

出　　处：《中国病理生理杂志》2012年第7期1326-1329,1334,共5页Chinese Journal of Pathophysiology

基　　金：广东省科技计划项目(No.2006B35502007;No.2010B031200013);广东省自然科学基金重点项目(No.04105351)

摘　　要：目的:建立N-甲基-N'-硝基-N-亚硝基胍(N-methyl-N'-nitro-N-nitrosoguanidine,MNNG)诱导的人端粒酶RNA组分(telomerase RNA component,TERC)缺陷的人支气管上皮细胞株(16HBE)恶性转化细胞模型。方法:将靶向TERC基因的shRNA干扰质粒载体转染16HBE细胞,G418抗性克隆筛选得到稳定转染的16HBE-1细胞,RT-PCR检测16HBE-1细胞TERC mRNA的干扰效率;用1 mg/L MNNG对16HBE-1细胞进行隔代染毒,每次染毒1 h;直到染毒27次转化灶的出现。分离扩增转化灶细胞并命名为16HBE-T,用软琼脂克隆形成实验和裸鼠成瘤实验鉴定细胞的转化程度。结果:从转化灶分离培养的细胞能在软琼脂中生长,且转化细胞能在裸鼠体内成瘤,HE染色后光镜下显示为鳞癌。结论:成功建立MNNG诱导的TERC基因缺陷的16HBE细胞恶性转化模型。AIM： To establish a malignant transformed human bronchial epithelial （16HBE） cell model in- duced by N - methyl - N - nitro - N - nitrosoguanidine （MNNG）. METHODS： A combination plasmid expressing shR- NA specific for the human telomerase RNA component （TERC） was transfected into 16HBE cells and G418 - resistant sta- ble clone was obtained, named 16HBE -1 cells. The mRNA expression of TERC in 16HBE -1 cells was detected by RT- PCR. 16HBE - 1 cells were treated with MNNG at concentration of 1 mg/L for 1 h in every other generation for 27 times until the transforming foci were formed. The cells from the transforming foci were separated and called 16HBE - T cells. Malignancy of 16HBE - T ceils was identified by colony formation in soft agar and tumorigenesis in nude mice. RESULTS ： 16HBE - T cells grew in soft agar and turned into tumor in nude mice. The tumor was squamous carcinoma confirmed by histopathological examination. CONCLUSION： The malignant transformation of TERC - deficient 16HBE cells is success- fully induced by MNNG.

关 键 词：端粒酶RNA 人支气管上皮细胞 RNA干扰 N-甲基-N’-硝基-N-亚硝基胍 

分 类 号：R363[医药卫生—病理学]