论文部分内容阅读
目的用小发卡RNA(small hairpin RNA,shRNA)干扰技术沉默N-Ras基因表达,探讨N-Ras基因表达对二羟环氧苯并芘(BPDE)转化人支气管上皮细胞(16HBE-T)恶性性状的影响。方法将已建立的针对N-Ras基因的shRNA干扰表达质粒载体pGPU6/GFP/Neo-NRas-566转染16HBE-T细胞,经G418筛选,建立稳定沉默N-Ras基因转化细胞系。采用Western blot筛选最佳N-Ras基因沉默细胞系,用流式检测N-Ras沉默细胞周期变化,用软琼脂实验和裸鼠成瘤实验进行细胞表型分析。结果成功培养出稳定沉默N-Ras基因的16HBE-T细胞系,Western blot显示N-Ras蛋白抑制率最高达86%。流式分析发现稳定沉默N-Ras基因表达诱导转化细胞G0/G1期捕获,软琼脂实验和裸鼠成瘤实验发现稳定沉默N-Ras基因降低了体外恶性转化细胞的集落形成率、抑制了体内肿瘤细胞生长。结论N-Ras基因沉默可以降低转化细胞的细胞周期进程和细胞恶性性状表型,提示N-Ras基因在二羟环氧苯并芘诱导细胞恶变过程中具有重要作用。
Objective To silence the expression of N-Ras gene by small hairpin RNA (shRNA) interference technique and explore the effect of N-Ras gene expression on malignant transformation of human bronchial epithelial cells (16HBE-T) induced by dihydroxyepoxybenzopyrene (BPDE) Impact. Methods 16HBE-T cells were transfected with the shRNA interference expression plasmid vector pGPU6 / GFP / Neo-NRas-566 targeting N-Ras gene and screened by G418 to establish a stable silencing N-Ras gene transformed cell line. The best N-Ras gene silencing cell line was screened by Western blot, the cell cycle was silenced by N-Ras flow cytometry, and the cell phenotype was analyzed by soft agar assay and nude mice tumorigenesis experiment. Results The 16HBE-T cell line with stable silencing of N-Ras gene was successfully established. Western blot showed that the inhibitory rate of N-Ras protein was up to 86%. Flow cytometry analysis showed that stable and silent N-Ras gene expression induced G0 / G1 arrest, soft agar assay and nude mouse tumorigenicity assay. Stable and silent N-Ras gene reduced the rate of colony formation in malignant transformed cells in vitro and inhibited the in vivo Tumor cell growth. Conclusion N-Ras gene silencing can reduce the cell cycle progression and the malignant phenotype of transformed cells, suggesting that N-Ras gene plays an important role in the carcinogenesis induced by dihydroxybenzophenone.