原儿茶酸对人胶质瘤细胞株U251MG增殖、凋亡的影响及其机制  

Effect of protocatechuic acid on the proliferation and apoptosis of human glioma cell line U251MG and the mechanism

作　　者：李宝龙[1] 杨大为[1] 刘阳[1] 李玉斌[1] 王同立 马智慧[1] 张海珢 张龙[1] 杨松海 Li Baolong;Yang Dawei;Liu Yang;Li Yubin;Wang Tongli;Ma Zhihui;Zhang Haiken;Zhang Long;Yang Songhai(Department of Neurosurgery,the First Hospital of Qinhuangdao,Qinhuangdao 066000,China)

机构地区：[1]秦皇岛市第一医院神经外科,066000

出　　处：《中华实验外科杂志》2021年第3期472-475,共4页Chinese Journal of Experimental Surgery

摘　　要：目的探讨原儿茶酸(PCA)对人胶质瘤细胞株U251MG增殖、凋亡的影响及相关分子机制。方法常规培养人胶质瘤细胞株U251MG。应用噻唑蓝(MTT)技术检测0、2.5、5.0、10.0、20.0μmol/L浓度PCA干预后各组细胞的活性。流式细胞术(FCM)检测PCA对U251MG细胞周期和凋亡的影响。应用实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)检测U251MG在药物干预前后增殖凋亡相关基因和的丝裂原活化蛋白激酶(MAPK)信号通路蛋白的变化,组间比较采用t检验。结果0μmol/L的PCA干预48 h后细胞活性为0.881±0.079、2.5μmol/L为0.757±0.077、5.0μmol/L为0.600±0.101、10.0μmol/L为0.532±0.056(F=35.131,P<0.01),差异有统计学意义。RT-qPCR和Western blot结果显示,随PCA浓度增加,增殖细胞核抗原(PCNA)的mRNA表达水平为1.172±0.153、0.756±0.059、0.601±0.061、0.390±0.059、0.236±0.036(F=55.618,P<0.01),差异有统计学意义;蛋白的表达水平分别为0.934±0.071、0.781±0.053、0.650±0.045、0.384±0.046、0.174±0.019(F=113.681,P<0.01),差异有统计学意义。FCM结果显示,PCA组U251MG细胞的G0/G1期比例[(71.033±8.570)%]高于对照组[(52.057±6.735)%,t=3.015,P<0.05],差异有统计学意义,G2/M比例[(18.833±7.639)%]低于对照组[(36.910±5.328)%,t=-3.362,P<0.05],差异有统计学意义;Western blot结果显示,增殖相关蛋白Cyclin A、Cyclin D1、Cyclin E表达在PCA组低于对照组(0.405±0.047比1.217±0.101、0.673±0.070比1.166±0.110、0.604±0.085比1.286±0.085,t=-12.625、-6.549、-9.827,P<0.01),而p21、p27表达在PCA组高于对照组(1.206±0.096比0.575±0.046、0.990±0.086比0.481±0.045,t=10.267、9.083,P<0.01)。FCM结果显示,PCA组U251MG细胞凋亡率(29.971±7.572)%高于对照组[(10.264±3.121)%,t=4.168,P<0.05];Western blot结果显示,凋亡相关蛋白bcl-2、pro-Caspase-3、pro-Caspase-9表达在PCA组低于对照组(0.337±0.024比1.198±0.113、0.430±0.044比1.150±0.098、0.486±0.049比1.278±0.107,t=-12.9Objective To explore the effect of protocatechuic acid(PCA)on the proliferation and apoptosis of human glioma cell line U251MG and related molecular mechanisms.Methods U251MG cells were routinely cultured.The thiazole blue(MTT)method was used to detect the cell viability of each group after the intervention of PCA at the concentrations of 0,2.5,5.0,10.0,20.0μmol/L.Flow cytometry(FCM)was used to detect the effect of PCA on the cell cycles and apoptosis of U251MG cells.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the changes of U251MG related genes and signalling pathway proteins before and after PCA intervention.Results MTT results showed that after 0,2.5,5,10,and 20μmol/L PCA was used for 48 h,the activities of U251MG cells were 0.881±0.079,0.757±0.077,0.600±0.101,0.532±0.056,respectively(F=35.131,P<0.01).RT-qPCR and Western blotting results showed that with the increase of PCA concentration,the expression levels of PCNA mRNA were 1.172±0.153,0.756±0.059,0.601±0.061,0.390±0.059,0.236±0.036(F=55.618,P<0.01),while levels of PCNA protein were 0.934±0.071,0.781±0.053,0.650±0.045,0.384±0.046,0.174±0.019(F=113.681,P<0.01).FCM results showed that the ratio of G0/G1 phase of U251MG cells in PCA group[(71.033±8.570)%]was higher than control group[(52.057±6.735)%,t=3.015,P=0.039],and the ratio of G2/M phase in PCA group[(18.833±7.639)%]was lower than control group[(36.910±5.328)%,t=-3.362,P=0.028];RT-qPCR and Western blotting results showed that the expression of proliferation-related proteins Cyclin A,Cyclin D1,and Cyclin E was lower in PCA group than in control group(0.405±0.047 vs.1.217±0.101,0.673±0.070 vs.1.166±0.110,0.604±0.085 vs.1.286±0.085,t=-12.625,-6.549,-9.827,P<0.01),and the expression of p21 and p27 was higher in PCA group than in control group(1.206±0.096 vs.0.575±0.046,0.990±0.086 vs.0.481±0.045,t=10.267,9.083,P<0.01).FCM results showed that the apoptosis rate of U251MG cells of PCA group was(29.971±7.5

关 键 词：胶质瘤 原儿茶酸 增殖 凋亡 信号通路 

分 类 号：R285.5[医药卫生—中药学]