过表达人骨保护素的乳腺癌细胞克隆的构建

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]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。

Creation Breast Cancer Cell Clone with Osteoprogegerin Over-expression

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.