Akt3稳定表达细胞株的建立及其对MDA-MB-231细胞增殖的影响

Effect of Stable Expression of Akt3 on Proliferation of MDA-MB-231 Cells

目的 构建人蛋白激酶Bγ（Akt3）基因编码区序列（cDNA）的真核表达载体、建立其稳定表达细胞株并观察其对MDA-MB-231细胞增殖的影响.方法 从流产胎儿脑组织中提取总RNA,采用RT-PCR方法扩增Akt3 cDNA的全长序列后克隆入pEGFP-N2质粒中,构建成Akt3基因真核表达载体,然后转染入MDA-MB-231细胞中,新霉素筛选稳定转染细胞克隆,通过MTT实验,研究转染Akt3基因前后细胞增殖的变化.结果 重组载体经酶切鉴定和测序证实目的 基因正确无误.Western印迹检测结果显示AKT3融合蛋白在MDA-MB-231细胞中表达良好,而转染空载体及未转染细胞对照中未见有此融合蛋白质条带;MTT结果显示AKT3表达上调的稳定克隆组,其增殖活性显著高于空载体稳定转染细胞组及未转染亲代细胞组,差异具有统计学意义（P＜0.01）,而后两者差异无统计学意义（P＞0.05）.结论 Akt3过表达可增强MDA-MB-231细胞的增殖.

Objective To construct eukaryotic expression vector pEGFP-N2-Akt3, to establish a cell clone stably expressing Akt3 and to study the effect of Akt3 on proliferation of breast cancer cells. Methods Total RNA was extracted from fetal brain tissue. Akt3 cDNA was amplified by RT- PCR, and inserted into pEGFP-N2 vector to construct pEGFP-N2-Akt3. The recombinant plasmid was then transfected into MDA-MB-231 cells by lipofectamine. The stable transfectants of MDA-MB- 231 cells were selected with G418. Western Blot was used to detect AKT3 expression level. MTT as- say were used to evaluate the effects of Akt3 on MDA-MB-231 cell proliferation. Results The re- combinant expression vector pEGFP-N2-Akt3 was successfully constructed. The expression of AKT3- EGFP fusion protein was only detected in MDA-MB-231 cells transfected with the Akt3 expression vector, but not in empty vector transfected or untransfected MDA-MB-231 cells. MTT assay showed that the proliferation of MDA-MB-231 cells with Akt3 stable expression was significantly higher than that of control cells （P 〈 0. 01 ） . Conclusion Akt3 can promote MDA-MB-231 cell proliferation.