【作者】 张祖斌；

【导师】 顾振纶； 蒋小岗； 周文轩； 郭次仪；

【作者基本信息】 苏州大学 ， 药理学， 2010， 硕士

【摘要】 目的:研究三氧化二砷(arsenic trioxide,As2O3)对人脑胶质瘤干祖细胞的抑制作用并探讨其机制。方法:MTT法检测不同浓度As2O3 (3, 6, 12μmo/L)对人脑胶质瘤干祖细胞增殖的影响; Hoechst33258荧光染色、透射电镜观察细胞形态变化;流式细胞仪(PI单染)分析细胞周期变化,流式细胞仪(Annein V/PI双染)检测细胞凋亡率;Western blot检测As2O3与人脑胶质瘤干祖细胞凋亡相关的MLK3/p-MLK3、c-jun/p-c-jun、Fas/FasL蛋白表达变化。结果:(1). MTT检测结果显示:3μmo/L As2O3作用人脑胶质瘤干祖细胞24 h,48 h,72 h后的抑制率分别为8.03%,9.96%和21.88%;6μmo/L As2O3作用人脑胶质瘤干祖细胞24 h,48 h,72 h后的抑制率分别为9.43 %,35.80 %和50.40 %;12μmo/L As2O3作用后的抑制率分别为21.88 %,45.47 %和63.10 %。24 h、48 h、72 h的IC50分别为:65.53μmol/L、10.65μmol/L和7.21μmol/L,提示As2O3对人脑胶质瘤干祖细胞的抑制作用呈浓度、时间依赖趋势。(2). Hoechst33258荧光染色结果显示:对照组细胞核大,Hoechst着色的细胞呈圆形,核内的荧光弱、亮度均一,呈淡兰色。As2O3给药组细胞染色质凝聚、细胞核边集、染色加深等细胞凋亡的形态学改变。透射电镜观察结果显示:对照组细胞膜表面微绒毛丰富,胞浆量少,细胞核大,核仁明显,核染色质电子密度低,染色质均一;6μmol/L As2O3给药48 h后,细胞膜不完整,细胞膜表面微绒毛和伪足减少,胞质内较多空泡,染色质浓染,细胞核边集,可见凋亡小体。(3).流式细胞仪凋亡率检测结果显示:0、6、12μmo/L As2O3作用人脑胶质瘤干祖细胞48 h后,凋亡率分别为3.1 %、12.07 %、29.53 %;作用72 h后,凋亡率分别为3.9 %、42.74 %、66.48 %,与对照组相比差异显著(P<0.05)。细胞周期检测结果:对照组G1期细胞占52.153 %,6μmo/L As2O3作用24 h,48 h,72 h后,G1细胞所占比例分别为64.184 %,74.790 %,88.467 %。,提示As2O3可使细胞周期阻滞在G1期。(4). Western blot检测结果:6μmo/L As2O3处理人脑胶质瘤干祖细胞12 h, 24 h和48 h之后,JNK/Fas信号通路相关蛋白MLK3/p-MLK3、c-jun/p-c-jun、Fas/FasL表达上调,3、6μmo/L As2O3处理48h之后,MLK3/p-MLK3、c-jun/p-c-jun蛋白表达也上调。结论:As2O3对人脑胶质瘤干祖细胞具有显著的抑制作用,该作用主要与诱导细胞凋亡并使细胞周期阻滞在G1期有关,激活JNK/Fas信号通路可能是其分子机制之一。更多还原

【Abstract】 Objective: To study the inhitory effect and its mechanism of arsenic trioxide on human glioma stem cells.Methods: Cell proliferation of As2O3 (3, 6, 12μmo/L) on human glioma stem cells was determined by MTT assay. The morphological changes were observed by Hoechst33258 fluorochrome staining and electron microscope; Cell cycle and apoptosis rate were analysed by flow cytometry. The levels of JNK pathway-mediated MLK3/p-MLK3, c-jun/p-c-jun and Fas/FasL involved in the effect of As2O3 on human glioma stem cells were studied by western blot.Results:(1). MTT assay showed that As2O3 had a significant anti-proliferation effect on human glioma stem cells in a dose-dependant and time-dependant manner. After treated with 6μmo/L As2O3 for 24 h, 48 h, 72 h the inhibition rates were 9.43 %,35.80 % and 50.40 %, respectively; After treated with 12μmo/L As2O3 for 24 h, 48 h, 72 h the inhibition rates were 21.88 %,45.47 % and 63.10 %, respectively.(2). Apoptosis morphological changes about chromatic agglutination and nuclear condensation were detected by Hoechst 33258 fluorochrome staining and electron microscope in As2O3 treated human glioma stem cells.(3). Apoptosis rates of human gioma stem cells treated with 0, 6, 12μmo/L As2O3 for 48 hours were 3.1 %, 12.07 %, 29.53 %, respectively; After trated with As2O3 for 72 h , the apoptosis rates were 3.9 %, 42.74 % and 66.48 % respectively (P<0.05). Cell cycle arrest was examined by Flow Cytometry (FCM). After treated with 6μmo/L As2O3 for 24h, 48h, 72h, the G1 phase rates were 64.184 %,74.790 %,88.467 %, respectively. It was 52.153 % in control group.(4). After treated with 6μmo/L As2O3 for 12h, 24h, 48h the JNK/Fas mediated proteins MLK3/p-MLK3, c-jun/p-c-jun and Fas/FasL were upregulated by western blot. When the cells were treated with 3、6μmo/L As2O3, the MLK3/p-MLK3, c-jun/p-c-jun proteins were also upregulated.Conclusion: The results suggested that As2O3 could inhibit the proliferation viability of human glioma stem cells (CD133+). It induced cell apoptosis and blocked the cell cycle at G1 phase. The molecule mechanisms may be associated with the activation of JNK/Fas pathway.更多还原