Fas/FasL信号途径参与17-羟-岩大戟内酯B诱导U251细胞凋亡

17-Hydroxy-jolkinolide B induced apoptosis of U251 cells via fas/fasL pathway

目的 :探讨Fas/Fas L信号转导途径在17-羟-岩大戟内酯B诱导人脑胶质瘤细胞U251细胞凋亡中的作用及机制。方法:以5-氟尿嘧啶(浓度为10μg/ml)为阳性对照。各组样品用四甲基偶氮唑蓝(MTT)法检测细胞增值抑制率;流式细胞仪Annexin V-FITC/PI检测细胞早期凋亡率;分光光度法检测Caspase-3和Caspase-8的相对活性;Western Blot法检测Fas和Fas L的蛋白表达。结果:与空白对照组相比,10μg/ml的17-羟-岩大戟内酯B对U251细胞的增殖没有显著性改变,但20μg/ml和40μg/ml的17-羟-岩大戟内酯B对U251细胞的增殖有显著性抑制作用,但40μg/ml的17-羟-岩大戟内酯B对U251细胞的增殖的抑制作用没有进一步改变,故应用20μg/ml 17-羟-岩大戟内酯B做后续试验。与单独应用20μg/ml 17-羟-岩大戟内酯B组相比,经0.1μg/ml、0.5μg/ml或1μg/ml抗Fas单克隆抗体预处理后,再加入20μg/ml17-羟-岩大戟内酯B时,U251细胞增值抑制率明显减弱,并选用0.5μg/ml抗Fas单克隆抗体进行后续实验。单独应用20μg/ml17-羟-岩大戟内酯B,细胞早期凋亡率、Caspase-3及Caspase-8相对活性较空白组明显升高,Fas及Fas L蛋白表达水平明显增强;而经0.5μg/ml抗Fas单克隆抗体预处理后再加入20μg/ml17-羟-岩大戟内酯B,可使细胞早期凋亡率、Caspase-3及Caspase-8的相对活性较单独应用20μg/ml17-羟-岩大戟内酯B明显下降,Fas及Fas L蛋白表达明显减弱。结论:17-羟-岩大戟内酯B可抑制U251细胞增殖和诱导细胞凋亡;Fas/Fas L信号通路介导了17-羟-岩大戟内酯B诱导的凋亡;Fas/Fas L通路活化导致了下游caspase途径的活化。

Objective： To study the effect and mechanism of the Fas/FasL pathway in 17-Hydroxy-jolkinolide B-induced apoptosis of U251 cells.Methods： Cell viability was measured by MTT.Annexin V-FITC was used to test the apoptosis rate.Absorption spectrometry was adopt to measure the relative activity of Caspase-3 and Caspase-8.Protein expressions of Fas and Fas L were detected by Western Blot.Results： Compared with the control group,the 17-Hydroxy-jolkinolide B 20μg/ml evidently inhibited the growth of U251 cells（ P 〈 0.05）,and the apoptosis rate of U251 cells,the relative activites of Caspase-3 or Caspase-8,and the expressions of Fas and Fas L were significantly increased,respectively（ P 〈 0.05）.Compared with 17-Hydroxy-jolkinolide B 20μg/ml group,the cell proliferation inhibition rate decreased in 17-Hydroxy-jolkinolide B 20μg/ml ＋ Anti Fas monoclonal antibody 0.1μg/ml group,0.5μg/ml group and 1μg/ml group,respectively（ P 〈 0.05）.The early apoptosis rate,the relative activity of Caspase-3 or Caspase-8 and the expression of Fas and Fas L decreased significantly in17-Hydroxy-jolkinolide B 20μg/ml ＋ Anti Fas monoclonal antibody 0.5μg/m L group,respectively（ P 〈 0.05）.Conclusion： The 17-Hydroxy-jolkinolide B evidently inhibits the growth of U251 and induces the apoptosis of U251; the mechanism of apoptosis-induced by 17-Hydroxy-jolkinolide B may be mediated by Fas/FasL pathways; the activation of Fas/FasL pathway leads to the activation of downstream caspase pathway.