摘要目的 探讨cyclin D1在宫颈上皮内瘤变及鳞状细胞癌中的表达及其与HPV16 E7基因表达的相关性.方法 SiHa、Hcc94细胞均为宫颈上皮来源的鳞癌细胞,其中Hcc94细胞的E6/E7基因缺失.采用即时荧光定量PCR、免疫组织化学法检测cyclin D1在子宫颈上皮内瘤变(CIN)及鳞状细胞癌组织中基因及蛋白的表达情况;采用逆转录PCR(RT-PCR)、Western blot、免疫组织化学方法检测cyclin D1在不同E7表达状态的鳞状细胞癌细胞系的表达情况;设计靶向HPV16 E7基因启动子区的siRNA序列,构建质粒转染SiHa细胞,筛选稳定沉默E7基因的细胞系命名为cm-16细胞株;将空质粒转染SiHa细胞,获得空质粒细胞系命名为B3细胞株.用RT-PCR和Western blot检测E7沉默cm-16细胞cyclin D1基因及蛋白表达情况.结果 cyclin D1仅在正常宫颈鳞状上皮副基底细胞中表达,在CIN1、CIN2、CIN3表达降低,在HPV阳性的宫颈鳞状细胞癌中阴性或少量点灶状表达;cyclin D1在E7阳性SiHa细胞中的表达水平较cm-16及Hcc94细胞减低(均P＜0.05).结论 E7高表达的子宫颈癌组织及细胞中cyclin D1低表达,HPV E7基因抑制子宫颈癌cyclin D1表达.

abstractsObjective To investigate the expression of cyclin D1 in cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma and its relationship with human papillomavirus 16 (HPV16) E7 gene expression.Methods Both SiHa and Hcc94 cell lines were obtained from cervical epithelial cells of squamous cell carcinoma.E6/E7 gene was silent in Hcc94 cell line.Expression levels of cyclin D1 mRNA and protein in CIN and squamous cell carcinoma were detected by QT-PCR and immunohistochemistry (IHC) respectively.SiRNA was constructed for targeting the promoter of HPV16 E7 and then transfected into SiHa cells to establish cm-16 line with stable silencing of E7.Control cell line B3 was obtained by blank plasmid transfection into SiHa cells.RT-PCR and Western blot were used to detect cyclin D1 mRNA and protein expression in the SiHa,B3,and cm-16 cells,respectively.Results Cyclin D1 was expressed in the basal cells of normal cervical squamous epithelia and the expression gradually decreased in the progression from CIN1 to CIN3.Squamous cell carcinoma showed negative or scattered expression of cyclin D1 (P ＜ 0.05).Both mRNA and protein of cyclin D1 in E7 + SiHa cells were lower than those in cm-16 and Hcc94 cells.Conclusion Squamous cell carcinoma with high HPV E7 expression shows low level of cyclin D1,suggesting that HPV16 E7 gene inhibits the expression of cyclin D1.