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目的 研究人乳头瘤病毒 (HPV) 16型 E6 / E7基因对人角质形成细胞增殖及细胞周期调控因子 p5 3,p Rb的影响 .方法 L ipofectamine介导法进行转染 ,筛选阳性克隆 ,免疫荧光染色检测 HPV16 E6 / E7在转染细胞内的表达 ;流式细胞仪分析细胞周期变化 ;免疫印迹分析 p5 3,p Rb表达情况 .结果 免疫荧光染色检测见转染细胞内有 HPVE6 / E7表达 ;流式细胞仪显示转染细胞 G1期细胞为 49.2 %比正常角质形成细胞(73.4% )少 ,S期细胞 (39.8% )比未转染细胞 (19.4% )增多 ,免疫印迹分析发现正常角质形成细胞和转染角质形成细胞 ,均在 p5 3,p Rb相应分子量处出现染色带 ,但转染细胞染色带的着色深度均明显弱于正常细胞 .结论 HPV16 E6 / E7能影响细胞的生长和增殖 ,其作用可能与 p5 3,p Rb有关
Objective To investigate the effect of human papillomavirus type 16 E6 / E7 gene on the proliferation of human keratinocytes and the effect of cell cycle regulators p53, pRb. Methods Transfection with L ipofectamine-mediated transfection, screening for positive clones, immunofluorescence The expression of HPV16 E6 / E7 in transfected cells was detected by staining. Changes of cell cycle were analyzed by flow cytometry. The expression of p53 and pRb was analyzed by Western blotting. Results The expression of HPVE6 / E7 in transfected cells was detected by immunofluorescence staining. Flow cytometry showed that G1 cells were 49.2% less transfected cells than normal keratinocytes (73.4%), S phase cells (39.8%) were more than non-transfected cells (19.4%), Western blot analysis found normal keratinocytes Both the cells and the transfected keratinocytes stained at the corresponding molecular weights of p53 and pRb, but the staining depth of the transfected cells was significantly weaker than that of normal cells. Conclusions HPV16 E6/E7 can affect cell growth and proliferation. , Its role may be related to p5 3, p Rb