重组东亚钳蝎镇痛抗肿瘤肽抑制人胆管癌细胞生长作用及其机制研究

Effects of recommbinant analgesic-antitumor peptide on the inhibition of cholangiocarcinoma cell line HuCC-T1

目的重组东亚钳蝎镇痛抗肿瘤肽(recommbinant analgesic-antitumor peptide,rAGAP)是从东亚钳蝎中提取纯化出的单体,已证明其具有明显的抗肿瘤活性。文中主要探讨rAGAP对体外培养的人胆管癌(cholangiocarcinoma,CCA)细胞株HuCC-T1的毒性作用,并探讨其抗肿瘤的机制。方法不同浓度的rAGAP刺激细胞,四甲基偶氮唑盐比色法(MTT法)检测细胞增长率,划痕愈合实验检测细胞迁移能力的变化;Hoechst荧光染色法和流式细胞术检测细胞凋亡率;采用Western blot方法检测凋亡相关蛋白Bcl-2和Bax的表达。结果①rAGAP可抑制HuCC-T1细胞的增殖和迁移,并呈时间和剂量依赖性。②Hoechst33342染色结果显示rAGAP处理组细胞形态发生改变,见细胞核染色质边集、核固缩;流式细胞术证实rAGAP可诱导HuCC-T1细胞凋亡。③Western blot结果显示:随着rAGAP浓度的增加,Bcl-2表达下调;Bax表达上调;而Bcl-2/Bax减小。结论 rAGAP可抑制CCA细胞HuCC-T1的生长与迁移,诱导细胞凋亡,其分子机制可能与Bcl-2/Bax信号通路有关。

Objective Recommbinant analgesic-antitumor peptide （rAGAP）, one Of scorpion toxic polypeptides extracted and purified from scorpionidae, has been showen to have significant antitumor activity. This study is to investigate the toxic effects and mecha- nism of rAGAP on cholangiocarcinoma cell line HuCC-T1 cultured in vitro. Methods HuCC-T1 cells were exposed to rAGAP with dif- ferent concentrations. Then cell growth rate was detected using MTI＇ method and the migration with wound healing assay; The apoptosis rate of cells were measured by Hoechst staining assay and flow cytometry （FCM） ; The apoptosis related protein level of Bcl-2 and Bax were analyzed by Western blot. Results ①rAGAP could inhibit the proliferation and migration of HuCC-T1 cells in a time-dose-de-pendent manner. ② Hoechst staining assay showed that cellular morphology changed and there were chromatin margination and karyopyknosis in the cell nucleus, when the cells were incubated with rAGAP, and FCM certified that rAGAP can induce apoptosis of HuCC-T1 ceils. ③Westem blot demonstrated that the expression of Bcl-2 was down-regulated and Bax up-regulated with increasing rAGAP concen- trations, so rAGAP could significantly decreases the production of Bcl-2/Bax. Conclusion rAGAP can inhibit the proliferation andmigration of HuCC-T1 cell line, and induce cell apoptosis. In the process of apoptosis, Bcl-2/Bax signal pathway may be involved.