NRP-1mAb对胃癌细胞BGC 823增殖的影响

目的 观察自主研发的抗NRP-1 b1／b2 IgG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、400 μg／ml NRP-1 mAb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。

Inhibitory effect of NRP-1mAb on the growth of gastric cancer cell BGC-823

Objective To observe the effect of NRP-1b1/b2 IgG monoclonal antibody (NRP-1mAb) 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 above 95％.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 therc 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.