抗人NRP-1单克隆抗体对肝癌HepG2细胞株的生长抑制作用及机制初探  

Inhibitory effect of anti-human NRP-1 monoclonal antibody on hepatocellular carcinoma cell line HepG2 and its mechanism in vitro

作　　者：谈文娟[1] 高春玲[1] 颜江华[2] 程小峰[1] 张蓓蓓[1] 王笑良[1] 

机构地区：[1]厦门大学附属成功医院放疗科,361003 [2]厦门大学医学院抗癌研究中心,361000

出　　处：《免疫学杂志》2016年第11期921-927,共7页Immunological Journal

基　　金：南京军区医学科技创新项目(12MA061;15MS104)

摘　　要：目的研究抗人神经纤毛蛋白-1(Neuropilin1,NRP-1)单克隆抗体对肝癌Hep G2细胞的生长抑制作用及其机制。方法小鼠腹水法制备抗NRP-1单克隆抗体(NRP-1 m Ab)并用r Protein A亲和柱纯化抗体,间接ELISA检测抗体的滴度水平。Western blot检测NRP-1 m Ab对Hep G2细胞的特异性,细胞免疫荧光和流式细胞术检测NRP-1蛋白在肝癌细胞株Hep G2上的表达,MTT法检测NRP-1 m Ab对Hep G2的生长抑制作用,Western blot检测ERK1/2、P-ERK1/2、Akt、P-Akt蛋白的表达水平。结果 SDS-PAGE和间接ELISA检测纯化的NRP-1 m Ab纯度为95%以上,效价为1×10^(-6);Western blot检测结果显示NRP-1 m Ab可与Hep G2细胞膜上的NRP-1蛋白特异性结合。细胞免疫荧光染色结果显示NRP-1定位于Hep G2细胞膜,流式细胞术结果显示NRP-1蛋白在Hep G2细胞上表达水平较高;MTT法检测结果显示NRP-1 m Ab对Hep G2细胞有生长抑制作用。Western blot检测到在不同浓度NRP-1 m Ab作用下,Hep G2细胞裂解液P-ERK1/2、P-Akt蛋白的条带信号逐渐减弱。结论纯化的NRP-1m Ab能抑制Hep G2细胞的生长,其抑制作用是通过EGF和HGF信号通路实现的。The aim of the experimental is to investigate the inhibitory effect of anti-human nerve cilia protein1（Neuropilin-1,NRP-1） monoclonal antibody（NRP-1 mAb） on hepatocellular carcinoma cell line HepG2 and its mechanism in vitro.Anti-human NRP-1 monoclonal antibody（NRP-1 mAb） was prepared from mouse ascites and purified by rProteinA affinity column assay.The titer of antibody was determined using indirect ELISA assay;the characteristic of NRP-1 mAb binding to NRP-1 was determined using Western blotting;the expression of NRP-1protein in hepatocellular carcinoma cell line HepG2 was determined using immunofluorescence assay and flow cytometry assay.Growth inhibition of HepG2 cells treated with different concentrations of NRP-1 mAb was determined using MTT assay,while Western blotting was used to detect the expression levels of ERK1/2,P-ERK1/2,Akt and P-Akt proteins.The results of SDS-PAGE and indirect ELISA showed that the purity of purified NRP-1mAb was more than 95%and the titer was 1×10^-6.Western blotting analysis suggested that NRP-1 mAb could bind specifically to NRP-1 on HepG2 cell;immunofluorescence staining showed that NRP-1 was located in the membrane of HepG2 cells.Flow cytometry analysis showed that the expression level of NRP-1 on HepG2 cell was relatively high.Western blotting analysis suggested that P-ERK1/2 and P-Akt expression levels were down-regulated after having incubated HepG2 cells with different concentrations of NRP-1 mAb.In conclusion,NRP-1 mAb could inhibit the growth of HepG2 cells（P〈0.05）,and its inhibitory effect is achieved by reducing the P-ERK1/2 and P-Akt expression.

关 键 词：人神经纤毛蛋白-1 肝癌 NRP-1单克隆抗体 生长抑制作用 

分 类 号：R735.7[医药卫生—肿瘤]