Annexin A2单克隆抗体抗肿瘤活性的实验研究

The antitumor activity of Annexin A2 monoclonal antibody

目的研究人膜联蛋白A2(Annexin A2)单克隆抗体的抗肿瘤活性,探讨Annexin A2在肿瘤发生发展中的作用。方法表达纯化Annexin A2重组蛋白,免疫小鼠及细胞融合制备单克隆抗体,用间接ELISA检测抗体的效价,Western blot和免疫共沉淀检测抗体的特异性。MTT法检测外源性Annexin A2抗体对肿瘤细胞生长的影响,流式细胞术检测其对肿瘤细胞周期的影响,RT-PCR及Quantitative real-time PCR检测对肿瘤细胞周期相关基因的影响,transwell检测其对HepG2细胞的侵袭及迁移的影响。结果 Annexin A2重组蛋白免疫小鼠,获得效价高的单克隆抗体。MTT结果显示,与对照细胞相比,外源抗体能够抑制HepG2细胞的生长。细胞周期相关检测结果显示,Annexin A2蛋白的表达与肿瘤细胞的生长周期相关基因具有相关性。transwell结果显示,外源抗体的处理抑制HepG2细胞的迁移,但促进了HepG2细胞的侵袭。结论 Annexin A2重组蛋白免疫Balb/c小鼠后,获得高滴度特异性的单克隆抗体。获得的Annexin A2单克隆抗体具有抑制生长迁移作用,为肿瘤的诊断及治疗提供了实验依据。

This study was performed to investigate the anti-tumor activity of human Annexin A2 monoclonal antibody. Annexin A2 recombinant protein was expressed and purified to prepare monoclonal antibodies by mouse immunization and cell fusion. The antibody titer was detected by indirect ELISA, and the specificity of the antibody was determined by Western blotting and immunoprecipitation. MTT assay and flow cytometry were used to detect the effect of exogenous Annexin A2 antibody on the growth and cycle of tumor cells, respectively. RT-PCR and quantitative real-time PCR were used to detect the effect of exogenous Annexin A2 monoclonal antibody on tumor cell cycle-related genes. The effects of the monoclonal antibody on invasion and migration of HepG2 cells were detected by transwell. Data showed that Annexin A2 monoclonal antibody with high titer was obtained from immunized mice;MTT results showed that compared with the control cells, the exogenous antibody could inhibit the growth of HepG2 cells. The results of cell cycle-related gene detection showed that the expression of Annexin A2 protein was correlated with the growth cycle-related genes of tumor cells;transwell results showed that the treatment with exogenous antibodies inhibited the migration of HepG2 cells, but promoted the invasion of HepG2 cells. Taken together, High-titer-specific monoclonal antibodies have obtained after immunizing Balb/c mice with Annexin A2 recombinant protein, which has the ability to inhibit growth and migration of tumor cells, providing experimental basis for the diagnosis and treatment of tumors.