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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 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(P<0.05)。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%,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(P<0.05)。结论 Rap1蛋白在卵巢浆液性癌组织中呈高表达并抑制细胞增殖。
Objective To investigate the expression of Rap1 protein in ovarian serous carcinoma and its role in the proliferation of serous carcinoma cells. Methods The expression of Rap1 in serous ovarian cancer tissues, benign ovarian tissues and normal ovarian tissues was detected by Western blot. Three Rap1 shRNA eukaryotic expression vectors were constructed by RNA interference technique. Three recombinant plasmids, empty plasmid, negative plasmid The recombinant plasmids were transfected into ovarian cancer cell line SKOV3 respectively. The recombinant plasmids were screened by Western blot. The cell lines stably transfected with SKOV3-Rap1-RNAi were screened by G418. The proliferation of SKOV3 cells with Rap1 expression was detected by MTT assay. Results Compared with benign ovarian tissues and normal ovarian tissues, Rap1 was highly expressed in serous ovarian cancer (P <0.05). Three recombinant plasmids pSUPER.retro.neo / GFP-Rap1i- # 1, -Rap1i- # 2 and -Rap1i- # 3 were successfully constructed by double enzyme digestion and DNA sequencing. Transient transfection indicated that the three plasmids Rap1 The efficiency of inhibition was 50%, 66%, 19% respectively. Finally, pSUPER.retro.neo / GFP-Rap1i- # 2 was transfected and the monoclonal cells Rap1i-1 and Rap1i-2 were screened by G418. (70%) and 48% (48%), respectively. Rap1i-1 cells were selected to detect the proliferation of SKOV3 cells with Rap1 expression deletion. The results showed that the proliferation of SKOV3 cells was stronger than that of the control group (P <0.05). Conclusion Rap1 protein is overexpressed in ovarian serous carcinoma and inhibits cell proliferation.