白藜芦醇对胶质瘤干细胞的辐射增敏作用及其机制的体内外研究
首发时间:2015-08-11
摘要:目的:本研究旨在从体内外探讨白藜芦醇(resveratrol,Res)对胶质瘤干细胞(GSCs)辐射敏感性的影响及其可能的机制。方法:体外采用集落形成实验分析resveratrol对GSCs辐射敏感性的影响。形态学观察和免疫荧光法检测干细胞特性。Western blot检测自噬相关蛋白LC-3和抗凋亡蛋白Bcl-2 的表达水平,透射电镜实验观察自噬泡的形成来检测自噬活性的变化。Annexin V-FITC/PI双染色法以及Hoechst 33258染色实验检测细胞的凋亡。彗星实验检测白藜芦醇对DNA损伤修复能力的影响。体内建立裸小鼠皮下移植瘤模型,绘制肿瘤生长曲线并采用Western blot、透射电镜实验进一步检测自噬和凋亡。结果:集落形成实验结果显示,白藜芦醇联合X射线可明显抑制GSCs生长(P<0.05)。形态学观察和免疫荧光结果显示,白藜芦醇联合X射线抑制GSCs的自我更新能力并诱导其分化。LC3表达情况和电镜实验观察双膜自噬泡的形成与分布情况都表明白藜芦醇增强了辐射诱导的自噬活性。Western blot分析Bcl-2蛋白表达、Annexin V-FITC/PI染色以及Hoechst 33258染色实验表明,白藜芦醇促进胶质瘤干细胞发生凋亡。彗星实验结果显示,白藜芦醇联合X射线削弱GSCs的DNA损伤修复能力。肿瘤生长曲线结果显示,白藜芦醇联合X射线可以显著抑制裸小鼠皮下移植瘤的生长(P<0.05);Western blot分析结果显示,白藜芦醇联合X射线治疗组中的LC3II /LC3I显著上调,抗凋亡蛋白Bcl-2表达水平明显下降(P<0.05);电镜结果显示,联合处理组中的双膜自噬泡显著增加,自噬被激活。结论:在体内外,白藜芦醇可以显著增强GSCs的辐射敏感性,并且可能是通过促进分化、增强辐射诱导的自噬活性、促进细胞凋亡以及削弱DNA损伤修复能力来协同发挥作用的。
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Radiosensitization effect and molecular mechanism of resveratrol on glioma stem cells in vitro and in vivo
Abstract:Aim: The aim of this study was to investigate the radiosensitization effect of resveratrol on glioma stem cells (GSCs) in vitro and in vivo and the molecular mechanisms involved. Methods: The clonogenic survival assay was performed to analyze the radiosensitization effect of resveratrol on GSCs. Cell morphology and immunofluorescence were used to detect the stemness of SU-2. Western blot and transmission electron microscopy were used to identify the changes of autophagy level. The apoptotic cells were observed using Hoechst 33258 staining and Annexin V-FITC/PI staining. Neutral comet assay was adopted to analyze the effect of resveratrol on repair of radiation induced DNA damage. The radiosensitization effect of resveratrol on GSCs in vivo was assessed by changes of the relative tumor volume(RTV). Results: Resveratrol and IR significantly reduced the colony formation capacity of GSCs and enhanced radiosensitivity as indicated by clonogenic survival assay. Cell morphology and immunofluorescence revealed that the administration of resveratrol and IR effectively inhibited self-renewal and induced the differentiation of GSCs. A combination of resveratrol with IR significantly increased autophagy and apoptosis level both in vitro cells and in nude mouse model as detected by western blot, transmission electron microscopy, Hoechst 33258 staining and Annexin V-FITC/PI staining. Finally, resveratrol significantly attenuated the repair of radiation induced DNA damage as reflected by the tail length of the comet. Conclusion: Our data demonstrate that the significant radiosensitization ability of resveratrol both in vitro GSCs and in vivo nude mouse model is attributed to its synergistic antitumor effects, including induction of differentiation and autophagy, promotion of apoptosis and prevention of DNA repair.
Keywords: Resveratrol GSCs radiosensitization autophagy apoptosis
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