海蛾活性部位抑制人宫颈癌HeLa细胞增殖及诱导凋亡作用研究

摘要

Aim To isolate HeLa cell proliferation-inhibitory active fraction from Pegasus laternarius Cuvier and explore its potential apoptosis-inducing mechanism.Methods To obtain the active fraction,the ethanol extract of Pegasus laternarius Cuvier was chromatographed by silica gel and sephadex LH-20 columns;MTT assay was used to evaluate the proliferation-inhibitory ability of active fraction on HeLa cells;AO/EB,PI and Annexin V-FITC/PI fluorescent staining flow cytometry were used to evaluate its apoptosis-inducing ability;the possible mechanism was investigated by analyzing the enzyme activity of caspase-3 and the protein expression of apoptosis-related genes in tumor cells.Results A fraction of C22 with high HeLa proliferation-inhibitory activity was isolated,with a yield of 0.73 ‰ and an IC50 of 36.3 mg · L-1;fraction C22 could increase the proportion of cells in sub-G0/G1 phase,phosphatidylserine eversion and other typical cell apoptosis in a dose-dependent manner;fraction C22 could down-regulate the expression of Bcl-2 and increase the enzyme of caspase-3 in HeLa cells.Conclusions The active fraction C22 from Pegasus laternarius Cuvier can inhibit the proliferation of HeLa cells by inducing apoptosis.The effect of inducing apoptosis may be conducted through mediating the mitochondrial Bcl2/caspase pathway.%目的 从中药材海蛾中分离具有抑制人宫颈癌HeLa细胞增殖活性的组分,并探讨其诱导凋亡的机制.方法 采用生物活性追踪法对海蛾乙醇浸提物进行硅胶柱层析、葡聚糖凝胶Sephadex LH-20柱层析分离抗肿瘤活性组分;采用MTT法评价其对肿瘤细胞的抑制作用;采用AO/EB、PI及Annexin V-FITC/PI荧光染色流式细胞术分析其诱导凋亡作用;通过分析肿瘤细胞中caspase-3活性及凋亡相关基因的蛋白表达水平,探讨其诱导凋亡的可能机制.结果 从海蛾中分离得到了具有较高HeLa细胞抑制活性的组分C22,得率为0.73‰,IC50为36.3 mg·L-1;组分C22作用于HeLa细胞后,可致细胞处于亚二倍期的比例增加、磷脂酰丝氨酸外翻等典型细胞凋亡现象,且具有剂量依赖性;组分C22可下调HeLa细胞Bcl-2的表达,降低胞内Bcl-2/Bax比例,增加caspase-3酶活性.结论 海蛾活性组分C22能诱导人宫颈癌HeLa细胞凋亡,抑制肿瘤细胞增殖,其诱导凋亡的机制可能是通过线粒体途径的Bcl-2/caspase通路发挥作用.