人参多糖介导Wnt/β-catenin信号转导诱导人鼻咽癌细胞CNE-2的凋亡  

Effect of ginseng polysaccharide-induced Wnt/β-catenin signal transduction pathway on apoptosis of human nasopharyngeal cancer cells CNE-2

作　　者：范家铭[1] 刘泽洪[2] 李静[2] 王亚平[2] 杨绿源[1] 黄江菊[1] 

机构地区：[1]重庆医科大学附属第一医院耳鼻咽喉头颈外科,重庆400016 [2]重庆医科大学组织学与胚胎学教研室干细胞与组织工程研究室,重庆400016

出　　处：《中国中药杂志》2013年第19期3332-3337,共6页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81171929;31271368);重庆市教委基因项目(KJ110308)

摘　　要：目的:观察人参多糖(ginseng polysaccharide,GPS)对人鼻咽癌细胞CNE-2的增殖和凋亡的影响以及可能的机制探讨。方法:CCK8法观察人参多糖对人鼻咽癌CNE-2细胞生长的影响。取对数生长期的CNE-2细胞,用不同质量浓度人参多糖(GPS)0,0.1,0.2,0.3,0.4 g·L-1分别作用48 h后,流式细胞术检测其对CNE-2细胞凋亡的诱导作用;Hoechst-33258染色和电镜观察细胞的形态改变。Real-time PCR检测细胞β-catenin mRNA的表达。采用免疫荧光染色检测细胞中β-catenin,TCF4蛋白定位及表达量的变化。Western blot检测细胞中β-catenin,Bcl-2,Bax蛋白的变化。结果:CCK8法测得人参多糖能明显抑制CNE-2细胞的增殖,其抑制作用呈剂量、时间依赖性,GPS处理细胞48 h的IC50为0.39 g·L-1;质量浓度分别为0.1,0.2,0.3,0.4 g·L-1的GPS作用人鼻咽癌CNE-2细胞48 h后细胞凋亡率分别为(5.69±0.29)%,(10.3±0.63)%,(15.4±0.74)%,(35.7±1.86)%,与对照组(2.08±0.11)%比较均有显著性差异(P<0.05);Hoechst-33258染色结果显示细胞凋亡特征。电镜下可见0.4 g·L-1GPS诱导48 h后可使CNE-2细胞出现凋亡小体。Real-time PCR显示β-catenin mRNA表达明显降低。激光共聚焦结果显示人参多糖作用48 h后β-catenin和TCF4在胞核中表达明显减少,β-catenin表达区域由胞核向胞膜转移。Western blot结果显示经浓度梯度增加的人参多糖作用CNE-2细胞48 h后,β-catenin和抗凋亡蛋白Bcl-2表达明显减弱;而促凋亡蛋白Bax表达上调(P<0.05)。结论:人参多糖可明显抑制CNE-2细胞增殖促进细胞凋亡。Wnt/β-catenin信号通路的受阻可能是GPS诱导人鼻咽癌细胞CNE-2凋亡的一个重要机制。Objective： To observe the effect of ginseng polysaccharide （GPS） on the proliferation and apoptosis of human na- sopharyngeal cancer cells CNE-2, and discuss the possible mechanism. Method： The effect of GPS on the growth of CNE-2 cells was observed by CCK8 assay. CNE-2 cells in the logarithmic phase were collected and processed respectively with different concentrations （0, 0. 1,0. 2, 0. 3, 0.4 g · L- 1 ） of GPS for 48 h. The flow cytometry was used to detect its induction effect on CNE-2 cell apoptosis. Hoechst-33258 cell staining and electron microscope were used to observe the morphological changes of cells. The/β-catenin mRNA ex- pression was detected by Real-time PCR. The protein localizations and expressions of/β-catenin and TCF4 were tested by the immuno- fluorescence staining. The expressions of/β-catenin, Bcl-2 and Bax proteins were detected by Western blot. Result： CCK8 assay re- sults showed that GPS could remarkably inhibit the proliferation of CNE-2 cells, with dose-time dependence. ICs0 of cells induced with GPS for 48 h was 0. 39 g · L- 1. After being processed with GPS with concentrations of 0. 1,0. 2, 0. 3, 0. 4 g · L-1 for 48 h, the cell apoptosis rates of human nasopharyngeal cancer cells CNE-2 were （ 5.69 ± 0. 29） %, （ 10. 3 ± 0. 63 ） %, （ 15.4 ± 0.74） % and （35.7 ± 1.86） %, respectively. Significant difference was observed compared with the control group （2. 08 ± 0. 11 ） % （ P 〈 0.05 ）. The results of Hoechst-33258 staining showed the characteristics of cell apoptosis. Under the electron microscope, apoptosis bodies could be observed among CNE-2 cells induced with GPS with the concentration of 0. 4 g· L-1 for 48 h. The results of Real-time PCR ／ showed a significant reduction in ~8-catenin mRNA expression. The results of laser confocal microscopy revealed notable decrease of fl-catenin and TCF4 expression in nucleus and transfer from nucleus to cell membranes in β-catenin expression areas after being pro- cessed with GPS for 48 h. Wester

关 键 词：人参多糖 CNE-2细胞 细胞增殖 凋亡 WNT Β-CATENIN 

分 类 号：R285.5[医药卫生—中药学]