胃癌血管靶向肽GX1抑制人胃癌血管内皮细胞增殖的机制研究

摘要

背景:为提高胃癌血管抑制治疗的选择性,本课题前期研究应用噬菌体呈现肽库技术筛选获得了人胃癌血管靶向性环七肽GX1,并发现GX1能抑制人胃癌血管内皮细胞增殖,诱导细胞凋亡.目的:探讨GX1抑制人胃癌血管内皮细胞增殖的相关机制.方法:分别以40 μmol/L GX1和40 μmol/L对照肽Pep2处理正常人脐静脉内皮细胞( HUVEC)和与人胃腺癌细胞株SGC7901体外共培养、产生肿瘤血管内皮相关特征的co-HUVEC.流式细胞术检测细胞周期分布,基因芯片检测筛选GX1组与对照肽Pep2组co-HUVEC差异表达基因并行GO分类,蛋白质印迹法检测凋亡相关基因蛋白表达.结果:与对照肽Pep2相比,GX1对co-HUVEC和正常HUVEC的细胞周期分布均无明显影响(P＞0.05).两组co-HUVEC共存在三百余个显著差异表达基因,其中与肿瘤生长、侵袭、转移相关者一百余个,功能主要涉及信号转导、凋亡、应激、细胞黏附、细胞代谢等.GX1组co-HUVEC的Bax、Cleaved Caspase-3蛋白表达明显上调,Bcl-2蛋白表达明显下调,Caspase-8蛋白表达无明显变化.结论:GX1可能通过下调Bcl-2/Bax比值,经线粒体途径诱导靶细胞凋亡,实现对人胃癌血管内皮细胞增殖的抑制作用.%For improving the selectivity of gastric cancer vascular inhibition therapy, we identified a human gastric cancer vasculature targeting cyclic 7-mer peptide ( GX1) by using phage-displayed peptide library technology. It was shown that GX1 could inhibit proliferation and induce apoptosis of human gastric cancer vascular endothelial cells. Aims; To investigate the possible mechanism of the inhibitory effect of GX1 on proliferation of human gastric cancer vascular endothelial cells. Methods; Human umbilical vein endothelial cells (HUVECs) with tumor endothelium characteristics (co-HUVEC) were generated by in vitro co-culturing with human gastric adenocarcinoma cell line SGC7901. Normal HUVEC and co-HUVEC were treated with 40 μmol/L GX1 and 40 μmol/L control peptide Pep2, respectively. The cell cycle distribution was assessed by flow cytometry, the differential expression genes in co-HUVEC between GX1 group and Pep2 group were analyzed by gene chip and categorized by gene ontology, and the protein expressions of apoptosis-related genes were determined by Western blotting. Results; Compared with control peptide Pep2, GX1 showed no significant effect on cell cycle distribution in both co-HUVEC and normal HUVEC {P >0.05). Gene chip analysis revealed that more than three hundred genes were notably differentially expressed in co-HUVEC between GX1 group and Pep2 group, among them, more than one hundred genes were associated with tumor proliferation, invasion and metastasis, including genes involved in signal transduction, apoptosis, stress, adherence and metabolism, etc. Western blotting confirmed the up-regulation of Bax and Cleaved Caspase-3 proteins, and the down-regulation of Bcl-2 protein in co-HUVEC treated with GX1, whereas no significant change of Caspase-8 protein was observed. Conclusions: GX1 may induce targeted cell apoptosis via decreasing the ratio of Bcl-2/Bax and activating the mitochondrial apoptotic pathway, finally leading to inhibition of proliferation of human gastric cancer vascular endothelial cells.