卵巢浆液性癌组织中Rap1蛋白表达检测及其对-第三军医大学学报.DOCVIP

卵巢浆液性癌组织中Rap1蛋白表达检测及其对浆液性癌细胞增殖的作用 肖正华1，2，高一萌1，彭慧娟１，张　瑜１，罗淑娟１，令狐华１*（400016重庆，重庆医科大学：附属第一医院妇产科１，附属永川医院妇产科２） [摘要] 目的 探讨卵巢浆液性癌组织中Rap1蛋白表达及其在浆液性癌细胞增殖中的作用。 方法 通过Western blot检测并比较Rap1在浆液性卵巢癌组织、卵巢良性组织和正常卵巢组织中的表达；应用RNA干扰技术构建3个Rapl shRNA真核表达载体，将3个重组质粒、空质粒、阴性质粒分别转染进卵巢癌细胞株 SKOV3，通过Western blot对重组质粒进行有效性筛选，G418筛选建立SKOV3-Rap1-RNAi 稳定转染细胞株；MTT法检测Rap1表达缺失的SKOV3细胞增殖能力的变化。 结果 与卵巢良性组织和正常卵巢组织相比，浆液性卵巢癌组织中Rap1呈现高表达（P0.05 ）。经双酶切和DNA测序证实成功构建了3个重组质粒pSUPER.retro.neo/GFP-Rap1i-#1，- Rap1i-#2，及- Rap1i-#3；瞬时转染提示上述3个质粒的Rap1 抑制效率分别是 50% 、66% 、19% ，遂最终采用pSUPER.retro.neo/GFP-Rap1i-#2 转染并用G418 筛选出单克隆细胞Rap1i-1和Rap1i-2，其Rap1蛋白抑制率分别为70% 和48% ，我们选取Rap1i-1细胞进一步检测Rap1表达缺失的SKOV3细胞增殖能力的变化，发现其增殖能力较对照组更强（P0.05）。 结论 Rap1蛋白对浆液性卵巢癌的增殖具有抑制作用。 [ 关键词 ] 浆液性卵巢癌；Rap1；肿瘤增殖 The detection of Rap1 expression and its role in the cell?proliferation?in serous ovarian cancer [Abstract] Objective To investigate the Rap1 expression and its role in the malignant growth in serous ovarian cancer. Methods Rap1 expression was evaluated through western blot analysis in serous EOC tissues, benign ovarianadenoma tissues and normal ovarian tissues. Three Rapl shRNA eukaryotic expression vectors were constructed with RNA interference technology. Three recombinant plasmids, empty vector and negative plasmid were transfected into ovarian cancer cell line SKOV3 through Lipofectamine 2000，then identified by restriction endonuclease digestion and DNA sequencing. The Rap1 siRNA efficiency was proved through monoclonal screening with G418 followed by western blot. Two strains of SKOV3-Rap1-RNAi cell lines were established. And malignant proliferation was evaluated by MTT. Results EOC tissues presented with significantly higher Rap1 expression than benign ovarian tumors and normal ovarian tissues （P0.05）. PSUPER.retro.neo/GFP-Rap1i-#2 was proved the most efficient through transient transfection. Accordingly, two monoclones of Rap1i cells ( Rap1i-1 and Rap1i-2 ) were established through pSUPER.retro.neo/GFP-Rap1i-#2 transfection followed