Inhibitory effect of NRP-1mAb on the growth of gastric cancer cell BGC-823
- 医学院－已发表论文 
目的观察自主研发的抗NRP-1b1/b2IgG单克隆抗体（NRP-1mAb）对胃癌BGC-823细胞生长的影响,并初步探讨可能的作用机制。方法实验室制备NRP-1mAb,采用SDS-PAGE检测纯度。采用0、25、100、200、400μg/ml NRP-1mAb培养胃癌BGC-823细胞,光学显微镜下观察细胞形态学变化;采用MTT法、集落形成实验、流式细胞术观察胃癌细胞BGC-823的增殖、集落形成和凋亡的情况;Western blotting法检测NRP-1mAb作用后Akt、p38、ERK、JNK信号蛋白磷酸化情况。结果SDS-PAGE检测显示,NRP-1mAb的纯度在95%以上。光学显微镜观察显示,NRP-1mAb作用后,BGC-823细胞形态呈凋亡改变。MTT实验显示,NRP-1mAb抑制BGC-823细胞的增殖作用呈浓度和时间依赖性（P〈0.01）。集落形成实验显示,不同浓度NRP-1mAb均能显著抑制BGC-823细胞的集落形成,其抑制率呈浓度依赖性（P〈0.01）。流式细胞术检测显示,不同浓度NRP-1mAb均明显促进BGC-823细胞凋亡,且大部分集中在早期凋亡。Western blotting检测显示,BGC-823细胞的Akt磷酸化受到抑制,ERK、p38、JNK的磷酸化水平无明显变化。结论NRP-1mAb能抑制胃癌细胞BGC-823的生长、促进凋亡,可能与抑制Akt磷酸化有关。Objective To observe the effect of NRP-1b1/b2 IgG monoclonal antibody（ NRP-1） on the growth of gastric cancer cell BGC-823,and to explore the possible mechanism of the antibody. Methods NRP-1mAb was prepared in laboratory,and the purity of antibody was detected by SDS-PAGE. Gastric cancer BGC-823 cells were cultured by 0,25,100,200,400 μg/ml NRP-1 mAb. The morphological changes of BGC-823 cells were observed by microscope. The proliferation,colony formation and apoptosis of gastric cancer BGC-823 cells were observed by MTT assay,colony forming assay and flow cytometry. The phosphorylation of related signal proteins was detected by Western blotting analysis. Results SDS-PAGE test showed that the purity of NRP-1mAb was above95%. Microscopy showed apoptotic changes of BGC-823 cells treated by NRP-1mAb. MTT assay showed that NRP-1mAb could inhibit the proliferation of BGC-823 cells in a time and dose dependent manner（ P〈0. 01）. Colony forming assay showed that different doses of NRP-1mAb could inhibit the colony formation of BGC-823 cells in a dose dependent manner（ P〈0. 01）. Flow cytometry showed that different doses of NRP-1mAb could promote the apoptosis of BGC-823 cells mainly at early apoptosis stage. It was found that the level of Akt phosphorylation was decreased after treated by NRP-1mAb,and there was no significant phosphorylation of ERK,p38 and JNK protein. Conclusion NRP-1mAb can inhibit the growth of gastric cancer cell BGC-823 and promote apoptosis,which may be related to the inhibition of Akt phosphorylation.