盐酸小檗碱诱导人脑胶质瘤细胞 U251凋亡及其与 HERG 钾离子通道的表达相关研究

摘要

目的：探讨盐酸小檗碱（ ber）诱导人脑胶质瘤细胞U251凋亡及其与HERG钾离子通道表达相关性。方法使用不同浓度的ber处理人胶质瘤细胞U251，采用四甲基偶氮唑盐（ MTT ）染色法测定细胞增值率，Hoechst33258荧光染色法观察细胞形态学改变，流式细胞术测定细胞凋亡率，分别采用实时荧光定量PCR与Western Blotting检测HERG钾通道的RNA及蛋白的表达情况。结果 MTT法检测显示细胞抑制率呈浓度与时间依赖性，且随药物浓度增加而增大（P＜0．05），其24 h的半数抑制浓度（IC50））是（8．78±0．20）μmol/l；Hoechst33258荧光染色法检测表明处理组出现典型的凋亡特征；且流式细胞术检测结果表明随着ber的剂量增加U251细胞的早期凋亡率及晚期凋亡率逐渐增大（P＜0．05）；RT-PCR与Western Bloting检测结果显示ber处理后U251细胞的HERG钾通道的RNA含量及蛋白的表达均上调，且随着药物浓度的增高而增高。结论在体外ber可诱导人胶质瘤细胞系U251的凋亡，并可能通过上调HERG蛋白的表达抑制U251增殖。%Objective To investigate the apoptosis effects and related expressions of HERG potassium channel of human glioma U 251 cell lines induced by berberine ( ber ) hydrochloride . Methods Human glioma U251 cells were treated with different concentrations of berberine hydrochloridein vitro , the cellular proliferation inhibition rate was Methyl thiazolyltetrazolium (MTT) assay, Hoechst33258 staining with fluorescent microscope was performed to observe the cell morphological changes of apoptosis , the apoptotic rate was detected by flow cytometry ( FCM ) , the both mRNA and protein expression of HERG were detected respectively by real -time fluorescent quantitative reverse transcription PCR and western blotting .Results The MTT assay discovered that the cellular proliferation showed on dose and time-dependent relationship , moreover with the concentration of it increased the apoptosis rate also increasing accordingly . The half maximal inhibitory concentration (IC50) was (8.78 ±0.20) μmol/l for 24 hours.Hoechst33258 fluorescent staining assay showed that the cell morphology of treated group revealed the typical characteristics of apoptosis , the early apoptotic rate and late apoptotic rate raised as dose of it by FCM .The both mRNA and protein expression of HERG which were detected respectively by Real -time fluorescent quantitative reverse transcription PCR and western blotting ,was gradually increased with dose of drug . Conclusion Berberine hydrochloride could induce apoptosis of U 251 cell in vitro , furthermore regulated up protein expression of HERG potassium to inhibit U 251 of the cellular proliferation .