Caspase-8和Bcl-2在脑肿瘤干细胞肿瘤坏死因子相关凋亡诱导配体耐药中的作用研究

 目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)对脑肿瘤干细胞生长的影响,研究Caspase-8和Bcl-2在肿瘤坏死因子相关凋亡诱导配体耐药中的作用。方法 应用CD133免疫磁珠分选法获得脑肿瘤干细胞,流式细胞术检测分选阳性率;亚神经球形成实验分析脑肿瘤干细胞的自我更新能力和增殖能力;四甲基偶氮唑盐法检测肿瘤坏死因子相关凋亡诱导配体对脑肿瘤干细胞生长的影响;Western blot法检测DR5、FADD、Caspase-8和Bcl-2蛋白的表达。结果 分选后CD133⁺细胞即脑肿瘤干细胞可达71%;脑肿瘤干细胞以神经球的方式生长;肿瘤坏死因子相关凋亡诱导配体作用前后,脑肿瘤干细胞都有极强的形成神经球的能力;肿瘤坏死因子相关凋亡诱导配体可以引起脑肿瘤干细胞死亡,加入Caspase-8或Caspases抑制剂,可明显阻断肿瘤坏死因子相关凋亡诱导配体所引起的细胞死亡(P<0.05);Western blot结果显示,脑肿瘤干细胞表达Caspase-8 蛋白水平降低,表达Bcl-2蛋白水平增加(P<0.05)。结论 脑肿瘤干细胞是脑肿瘤对肿瘤坏死因子相关凋亡诱导配体耐药的根源,Caspase-8蛋白表达减少及Bcl-2蛋白表达增强使Caspase-8不能活化,导致了脑肿瘤干细胞发生肿瘤坏死因子相关凋亡诱导配体耐药。

Research of Caspase-8 and Bcl-2 on TRAIL Resistance in Brain Tumor Stem Cells

OBJECTIVE To detect the effect of TRAIL on the growth of brain tumor stem cells, study the role of Caspase-8 and Bcl-2 of TRAIL resistance. METHODS Brain tumor stem cells were isolated by CD133 magnetic sorting, and the positive rates of CD133^＋cells were detected by flow cytometry, the self-renewing capicity of brain tumor stem cells was examined by subneurosphere formation assay, and the percentage of cell death were examined by MTS assay, the expression of DR5, FADD, Caspase-8 and Bcl-2 proteins was detected by Western blot. RESULTS The positive rates of CD133^＋cells were 71% after CD133 magnetic sorting. Brain tumor stem cells grow as neurosheres and have the abilities to reform neuroshperes, the capacity of neurosphere formation was significant increased after TRAIL treatment or not. Brain tumor stem cells were dead after TRAIL treatments, Caspase-8 and Caspases inhibitor can block the cell death that induced by TRAIL(P<0.05). The expression of Caspase-8 protein was decreased and Bcl-2 protein was increased in brain tumor stem cells(P<0.05). CONCLUSION Brain tumor stem cells may response to TRAIL resistance, the reason is the lower expression of Caspase-8 protein and over expression of Bcl-2 protein which is unable to activate Caspase-8.