Anxa5真核表达质粒构建及其对小鼠肝癌Hca—P细胞增殖影响观察

目的：构建pcDNA3。1-V5／HisB—Anxa5真核表达质粒，获得膜联蛋白A5（AnnexinA5，Anxa5）表达稳定上调的小鼠肝癌Hca—P细胞株，探究Anxa5上调对Hca—P增殖的影响。方法：利用RT—PCR扩增小鼠全长Anxa5编码序列，将其克隆到pcDNA3．1-V5／HisB载体中，并将构建的pcDNA3．1-V5／HisB-Anxa5表达质粒转入Hca—P细胞。采用（；418筛选及有限稀释法获得单克隆细胞株，观察细胞形态变化。蛋白质印迹法分析Anxa5表达情况，CCK-8法检测相应Hca—P细胞的增殖能力。结果：酶切及测序结果显示，pMD R 18-T—Anxa5克隆质粒和pcDNA3．1-V5／HisB-Anxa5表达质粒构建成功；获得了Anxa5表达稳定上调的单克隆细胞株；与正常Hca-P比较，Anxa5蛋白在pcDNA3．1一V5／HisB-Anxa5-Hca-P单克隆细胞中上调了59．5％，P一0．016；pcDNA3．1-V5／HisB～Anxa5-Hca—P增殖显著加快，P=0．002。结论：成功构建了Anxa5真核表达质粒并获得Anxa5表达稳定上调的小鼠肝癌Hca—P细胞株，Anxa5上调对Hca—P细胞的增殖具有促进作用，为后续研究提供实验基础。

Construction of Anxa5 eukaryotic expression plasmid and its effect on cell proliferation of mouse hepatocarcinoma cell line Hca-P

OBJECTIVE：To construct eukaryotic expression plasmid of pcDNA3.1-V5/HisB-Anxa5.obtain the Hea- P monoclonaI cell line with the stable up-regulation of annexin A5 （Anxa5） and investigate the effect of Anxa5 up-regula tion on cell proliferation. METHODS： The full-length coding sequence of mouse Anxa5 was amplified by using standard RT-PCR. The amplified Anxa5 was cloned into pcDNA3. 1-V5/HisB vector and the constructed peDNA3. 1-V5/ HisB-Anxa5 plasmid was transfected into Hca P cells. The monoclonal Hca-P cell with stable overexpression of Anxa5 was obtained by G418 screening and limited dilution. The cell morphology changes were observed. Western blotting was performed to measure the expression level of Anxa5. Cell proliferation ability was determined by CCK-8 assay. RESULTS： The cloning plasmid pMD~18-T-Anxa5 and eukaryotic expression plasmid pcDNA3.1-V5/HisB-Anxa5 were successfully constructed. The monoclonal Hca-P cell lines with Anxa5 stable overexpression were obtained. Compared with normal Hca-P cells, Anxa5 protein expression level in pcDNA3. 1-VS/HisB-Anxa5-Hca-P was up-regulated by 59. 5% （P = 0. 016）, proliferation of peDNA3.1-VS/HisB-AnxaS-Hca-P was increased significantly （ P = 0. 002 ）. CONCLUSIONS： Anxa5 eukaryotic expression plasmid was successfully constructed. The monoclonal Hca-P cell lines with Anxa5 stable overexpression were obtained and Anxa5 up-regulation facilitates the proliferation of HcwP cell.