过表达人骨保护素的乳腺癌细胞克隆的构建.docVIP

过表达人骨保护素的乳腺癌细胞克隆的构建建议修改 建议修改 张帆 周艳 唐鹏 姜军 第三军医大学西南医院乳腺中心，重庆400038 摘要： 目的 建立过表达人骨保护素（Osteoprotegerin，OPG）的MDA-MB-231乳腺癌细胞克隆。方法　从pOTB7- OPG 上获得OPG基因片段并用PCR方法扩增，将其连接于真核表达质粒pIRES2-EGFP，酶切鉴定及测序后，转染MDA-MB-231细胞，以G418 加压筛选，对转染细胞行单克隆化，稳定转染细胞行RT－PCR和Western blot 检测，确定其OPG mRNA 和蛋白表达情况，MTT法检测过表达OPG对MDA-MB-231细胞生长速率的影响。结果 成功构建了pIRES2-EGFP-OPG重组质粒；稳定转染细胞的OPG mRNA 和蛋白表达均较对照升高；过表达OPG对MDA-MB-231细胞生长速率无明显影响。结论 本研究成功建立了过表达OPG的MDA-MB-231细胞克隆，为进一步深入探讨肿瘤细胞自身OPG表达在乳腺癌骨转移发生发展中的作用提供相关实验基础。 关键词：乳腺癌；骨保护素；过表达；MDA-MB-231细胞；pIRES2-EGFP Construction breast cancer cell clone with osteoprotegerin over-expression ZHANG Fan, ZHOU Yan, TANG Peng, JIANG Jun (Breast Disease Center, Southwest Hospital , Third Military Medical University , Chongqing 400038 , China) Abstract: Objective To construct MDA-MB-231 breast cancer cell clone with osteoprotegerin(OPG) over-expression. Methods: To construct the recombinant plasmid pIRES2-EGFP-OPG, full length OPG cDNA with BglII and EcoRI amplified by PCR was digested and inserted into eukaryotic expression plasmid pIRES2-EGFP. After identification of restriction endonuclease and sequencing, the recombinant plasmid and empty plasmid were transfected into MDA-MB-231 cells by Lipofectamine TM 2000, respectively. The stably transfected clones after G418 screening were identified with RT－PCR and Western blot. The different growth rates of MDA-MB-231 cells with OPG over-expression and normal expression were detected by MTT. Results: The eukaryotic expression plasmid pIRES2-EGFP was constructed successfully. OPG mRNA and the protein level of MDA-MB-231 cell clone stably transfected with pIRES2-EGFP-OPG were higher than those of the stably transfected pIRES2-EGFP. OPG over-expression did not change the growth rate of MDA-MB-231 cells. Conclusion The breast cancer cell clone with OPG over-expression was constructed successfully in our study. This can provide a related experimental basis for further exploring the role of OPG expression by tumor cell itself in the dev