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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。
Objective To study the inhibitory effect of anti-human neuropilin 1 (NRP-1) monoclonal antibody on Hep G2 cell growth and its mechanism. Methods The anti-NRP-1 monoclonal antibody (NRP-1 m Ab) was prepared by ascites method in mice and the antibody was purified by r Protein A affinity column. The antibody titers were detected by indirect ELISA. Western blot was used to detect the specificity of NRP-1 m Ab in Hep G2 cells. The expression of NRP-1 protein was detected by flow cytometry and immunocytochemistry. The expression of NRP-1 m Ab was detected by MTT assay. . The expression of ERK1 / 2, P-ERK1 / 2, Akt and P-Akt protein were detected by Western blot. Results The purity of NRP-1 m Ab purified by SDS-PAGE and indirect ELISA was above 95% with a titer of 1 × 10 ~ (-6). Western blot results showed that NRP-1 m Ab could bind to Hep G2 cell membrane NRP-1 protein. The results of immunofluorescence staining showed that NRP-1 localized on the membrane of Hep G2 cells. The results of flow cytometry showed that NRP-1 protein was highly expressed on Hep G2 cells. MTT assay showed that NRP-1 m Ab Growth inhibition. Western blot analysis showed that the band signals of P-ERK1 / 2 and P-Akt in Hep G2 cell lysate decreased gradually with different concentrations of NRP-1 m Ab. Conclusion The purified NRP-1m Ab can inhibit the growth of Hep G2 cells, and its inhibitory effect is through the EGF and HGF signaling pathways.