姜黄素通过Wnt/β-catenin信号通路调控胶质瘤细胞增殖和凋亡的实验研究  

Curcumin regulation of glioma cell proliferation and apoptosis through Wnt/β-catenin signaling pathway： an experimental study

作　　者：马光涛[1] 张燕[2] 李晨龙[3] 郄福忠[1] 曹振宇[1] 蒋传路[3] 

机构地区：[1] 大庆油田总医院神经外科,大庆163411 [2] 大庆油田总医院心电室,大庆163411 [3]哈尔滨医科大学附属第二医院神经外科

出　　处：《中华神经外科杂志》2015年第6期625-630,共6页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金（81372700）

摘　　要：目的 探讨姜黄素通过Wnt/β-catenin信号通路对胶质瘤细胞的增殖及凋亡影响.方法 研究对象为人胶质母细胞瘤U251细胞系,实验分为空白对照组和姜黄素实验组（实验浓度分别为10 μmol/L和20 μmol/L）.细胞毒性实验研究姜黄素对U251胶质瘤的细胞毒性作用.四甲基偶氮唑蓝（MTT）比色法观察增殖能力;Caspase-3活性检测及流式细胞仪（FCM）实验检测细胞凋亡;流式细胞术检测细胞周期;实时定量PCR、Western-blot和免疫荧光检测法检测细胞内的β-catenin、c-myc表达水平.荧光素酶实验检测姜黄素作用后Wnt/β-catenin通路活性改变.结果 随着姜黄素作用浓度的增加,U251胶质瘤细胞的细胞毒性增强,在姜黄素实验组中,当浓度为5、10、20、和40 μmol/L时,细胞活性分别为（88.55±2.35）％、（62.78 ±3.35）％、（35.17±2.05）％和（7.47±1.92）％（P＜0.05）,并且增殖抑制作用明显增强,测得姜黄素作用24h的IC50为16.32 μmol/L,故姜黄素实验组浓度选用10 μmol/L和20 μmol/L;实验组（10μmol/L和20 μmol/L） Caspase-3的相对活性分别为（3.64±1.49）％,（5.41±0.36）％,与对照组（0.91±0.62）％相比,明显增强（P＜0.05）;流式细胞术检测姜黄素作用48 h后,实验组U251细胞凋亡率分别为（14.7±2.21）％和（35.6±1.37）％,与对照组凋亡率（0.5％±0.67）％相比,差异具有统计学意义（P＜0.05）.流式细胞仪检测姜黄素可以阻滞细胞周期于G2/M期,对照组,及姜黄素实验组（10 μmol/L和20 μμmol/L）的G2/M期分别为（7.52±3.84）％,（16.28±2.73）％,（25.47±5.61）％（P＜0.05）;姜黄素作用于胶质瘤后β-catenin的mRNA水平下降,荧光定量PCR检测结果为（93.87±5.73）％,（68.83±7.67）％和（47.46±8.52）％（P＜0.05）,差异具有统计学意义.Western-blot实验显示β-catenin和c-myc蛋白表达水平均下降;免疫荧光实验发现姜黄素可以抑制β-catenin进入细胞核,同时减�Objective To investigate the effects of curcumin on glioma cell proliferation and apoptosis through Wnt/β-catenin signaling pathway.Methods The research objects were the human glioblastoma U251 cell lines.The experiment was divided into either a blank control group or a curcumin experiment group （the experiment concentrations were 10 μmol/L and 20 μmol/L respectively）.The cell toxicity test was used to study cellular toxic effect of curcumin on U251 glioma.Four methyl thiazolyl tetrazolium （MTT） assay was used to observe the cell proliferative capacity; the caspase-3 activity assay and flow cytometry （FCM） test were used to detect cell apoptosis ; flow cytometry was used detect the cell cycle;real-time quantitative PCR,Western-blotting,and immunofluorescence assay were used to detect the expression levels of intracellular β-catenin and c-myc.The luciferase assay was used to detect the changes of Wnt/β-catenin signaling pathway activity after the effects of curcumin.Results With the increased effect concentration of curcumin,the cytotoxicity of U251 glioma cells enhanced.In the curcumin experimental group,when the concentrations were 5,10,20,and 40 μmol/L,the cells activities were 88.55% ±2.35%,62.78% ±3.35%,35.17% ±2.05%,and 7.47% ± 1.92%,respectively （P 〈0.05）,and the effect of proliferation inhibition enhanced obviously.IC50 of the measured curcumin effect for 24 h was 16.32 μmol/L,therefore,the concentrations of 10 μmo]/L and 20 μmol/L in the curcumin experimental group were chosen; the relative activities of caspase-3 in the experimental group （10 μmol/L and 20 μmol/L） were 3.64% ± 1.49% and 5.41% ±0.36% respectively.Compared with the control group 0.91% ± 0.62%,the activities enhanced significantly （P 〈 0.05）.After flow cytometry was used to detect the curcumin effects for 48 h,the U87 apoptotic rates of the experimental group were 14.7% ± 2.21% and 35.6% ± 1.37% respectively.Compared with the apoptotic rate of the control group （0.5% ±0.67%）,there was sig

关 键 词：神经胶质瘤 姜黄素 WNT/Β-CATENIN通路 细胞增殖 细胞凋亡 

分 类 号：R285[医药卫生—中药学]