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目的:构建pcDNA3.1-V5/HisB-Anxa5真核表达质粒,获得膜联蛋白A5(Annexin A5,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G418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○R18-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-V5/HisB-Anxa5-Hca-P单克隆细胞中上调了59.5%,P=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。
OBJECTIVE: To construct eukaryotic expression vector pcDNA3.1-V5 / HisB-Anxa5 and obtain Hca-P cell line with stable up-regulated expression of Annexin A5 (Anxa5) in mice to investigate the effect of Anxa5 upregulation on the proliferation of Hca-P influences. Methods: The full-length mouse Anxa5 coding sequence was amplified by RT-PCR and cloned into pcDNA3.1-V5 / HisB vector. The recombinant plasmid pcDNA3.1-V5 / HisB-Anxa5 was transformed into Hca-P cell. Monoclonal cell lines were obtained by G418 screening and limited dilution method. Morphological changes were observed. The expression of Anxa5 was analyzed by Western blotting. The proliferation of Hca-P cells was detected by CCK-8. Results: The results of restriction enzyme digestion and sequencing showed that the recombinant plasmid pMD ○ R18-T-Anxa5 and pcDNA3.1-V5 / HisB-Anxa5 were constructed successfully. The monoclonal cell line with stable expression of Anxa5 was obtained. Compared with normal Hca-P Anxa5 protein was up-regulated 59.5% in pcDNA3.1-V5 / HisB-Anxa5-Hca-P monoclonal cells, P = 0.016. The proliferation of pcDNA3.1-V5 / HisB-Anxa5-Hca- 0.002. CONCLUSION: Anxa5 eukaryotic expression plasmid was successfully constructed and HCC-P cell line of mouse hepatoma with stable expression of Anxa5 was obtained. Upregulation of Anxa5 could promote the proliferation of Hca-P cells and provide the experimental basis for further study.