MNNG诱导TERC基因沉默的16HBE细胞恶性转化模型的建立

目的: 建立N-甲基em>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。

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

AIM: To establish a malignant transformed human bronchial epithelial(16HBE) cell model induced by N-methyl-N’-nitro-N-nitrosoguanidine(MNNG).METHODS: A combination plasmid expressing shRNA specific for the human telomerase RNA component(TERC) was transfected into 16HBE cells and G418-resistant stable 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 cells 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 successfully induced by MNNG.