阻断Eag-1通道活性抑制胶质瘤细胞增殖的研究

目的 探讨阻断Eag-1通道活性对胶质瘤细胞生物活性的影响. 方法 设计3对Eag-1通道蛋白的特异性小分子干扰RNA(siRNA)并转染入U87细胞,RT-PCR及Western blotting检测其对U87细胞Eag-1 mRNA和蛋白表达的影响;将50 nmol/L siRNA1、siRNA2转染U87细胞,同时设5、10、20、30和40mmol/L奎尼丁(Eag-1通道特异性阻断剂)组和空白对照组,MTT法观察作用24、48、72 h后U87细胞增殖情况,流式细胞仪检测作用48 h后细胞周期、细胞凋亡率、胞内活性氧簇(ROS)浓度的变化. 结果 RT-PCR及Western blotting结果 显示siRNA1和siRNA2转染U87细胞12 h后细胞Eag-1 mRNA、蛋白的表达产物带明显弱于空白对照组,而siRNA3转染组产物条带虽也弱于空白对照组,但条带仍清晰;MTT检测结果 显示与空白对照组比较,50hmol/LsiRNA1、siRNA2转染组和10、20、30、40 mmol/L奎尼丁组细胞培养24、48和72 h后吸光度值均降低,差异有统计学意义(P＜0.05),奎尼丁IC50为33.7mmol/L.流式细胞仪分析显示与空白对照组比较,50nmol/L siRNA1、siRNA2转染组和33.7mmol/L奎尼丁组G1期细胞百分比、细胞凋亡率和胞内ROS水平均增加,差异有统计学意义(P＜0.05). 结论 阻断Eag-1通道活性能明显抑制胶质瘤细胞增殖,使G1期细胞比例、胞内ROS水平明显升高并诱导其凋亡.

Eag-1 channel blocking inhibits the proliferation of glioma cells

Objective To evaluate the influence of Eag-1 channel blocking on bioactivity of glioma cells in vitro. Methods Different small interfering RNAs (siRNAs) targeting for Eag-1 channel were designed and transfected to the U87 cells, and the blocking effects of those siRNAs were further confirmed on mRNA and protein levels by RT-PCR and Western blotting. The 50 nmol/l siRNAs (siRNA1 and siRNA2) and quinidine (5, 10, 20, 30 and 40 mmol/l) were used to block the activity of Eag-1 channel, respectively; and blank control group was also established. The proliferation of U87 cells 24, 48 and 72 h after the treatments was detected by MTT method; the changes of generation cycle,apoptosis ratio and intracellular reactive oxygen species (ROS) concentration were detected by flow cytometry. Results High mRNA and protein levels of Eag-1 channel on glioma cell line U87 were confirmed in the blank control group, however, siRNA1 and siRNA2 transfection groups showed significantly lower mRNA and protein levels of Eag-1 channel on glioma cell line U87. MTT method indicated that, 24, 48 and 72 h after the treatments, the proliferation of U87 cells in the siRNA1 and siRNA2 transfection groups, and quinidine treatment groups (10, 20, 30 and 40 mmol/l) was significantly inhibited as compared with that in the blank control group (P＜0.05). The IC50 value of quinidine is33.7mmol/l. As compared with the blank control group, 50 nmol/L siRNA1 and siRNA2 transfection groups, and 33.7 mmol/l quinidine treatment group enjoyed a significantly increased cell percentage at G1 stage, cell apoptosis ratio and intracellular ROS level (P＜0.05). Conclusion Eag-1 channel blocking can obviously inhibit the proliferation of glioma cells, increase the cell percentage at G1 stage and intracellular ROS level, and induce apoptosis of glioma cells.