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[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。
[Objective] To establish a clone of MDA-MB-231 breast cancer cells overexpressing human osteoprotegerin (OPG). [Method] The OPG gene fragment was obtained from pOTB7-OPG and amplified by PCR. The fragment was ligated to the eukaryotic expression plasmid pIRES2-EGFP. After identification and sequencing by enzyme digestion, the MDA-MB-231 cells were transfected with GFP The expression of OPG mRNA and protein was detected by RT-PCR and Western blotting. The effect of overexpression of OPG on the growth of MDA-MB-231 cells was detected by MTT assay. [Results] The recombinant plasmid pIRES2-EGFP-OPG was successfully constructed. The expression of OPG mRNA and protein in stable transfected cells was higher than that in control cells. Overexpression of OPG had no significant effect on the growth of MDA-MB-231 cells. [Conclusion] The clone of MDA-MB-231 cells overexpressing OPG was successfully established, which provided the experimental basis for further study on the role of OPG expression in tumor cells in the development of bone metastasis in breast cancer.