过表达人骨保护素的乳腺癌细胞克隆的构建  

Creation Breast Cancer Cell Clone with Osteoprogegerin Over-expression

作　　者：张帆[1] 周艳[1] 唐鹏[1] 姜军[1] 

机构地区：[1]第三军医大学西南医院,重庆400038

出　　处：《中国肿瘤》2008年第11期981-984,共4页China Cancer

基　　金：国家自然科学基金(30700814);第三军医大学2006年度中青年科研基金

摘　　要：[目的]建立过表达人骨保护素(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Westernblot检测,确定其OPGmRNA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OPGmRNA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]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实验基础。[Purpose] 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 Bgl Ⅱ and EcoR Ⅰ 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-OPG 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. This can provide a related experimental basis for further exploring the role of OPG expression by tumor cell itself in the development and progression of breast cancer bone metastasis.

关 键 词：乳腺肿瘤 骨保护素 MDA—MB-231细胞 质粒 

分 类 号：R73-3[医药卫生—肿瘤] R737.9[医药卫生—临床医学]