外泌体介导的细胞通讯在维持恶性胶质瘤干细胞动态平衡中的作用

The purpose of this grant is to investigate the roles of cell-cell communication mediated by exosomes released from glioblastoma multiforme (GBM) stem cells on maintenance of the dynamic equilibrium of GBM stem cells and their mechanism of activity. GBM stem cells will be enriched with cytokines and serum free medium, exosomes released from GBM stem cells will be isolated and purified with ultracentrifugation and ExoQuick kit; the adherent monolayer GBM cells and GBM bearing mice will be treated with the isolated exosomes; By cell biological and molecular biological protocols the quantity and component of exosomes released from both GBM stem cells and the adherent monolayer GBM cells will be studied; the effects of exosomes released from GBM stem cells on the stemness phenotype, tumorigenicity and GBM stem cell subpopulation change of the adherent monolayer GBM cells will be investigated in vitro and in vivo. The findings of this project will be helpful to elucidate the roles, factors and mechanisms of exosomes released from GBM stem cells in maintaining the dynamic equilibrium of GBM stem cells, and provide theoretical bases and scientific evidence for researching and developing pharmaceutical agents and exosome encapsulated drugs to target at both cancer stem cells and differentiated non stem cancer cells, blocking cancer stem cells from releasing exosomes, preventing non stem cancer cells from dedifferentiation and transforming into cancer stem cells for destroying the dynamic equilibrium of cancer stem cells, eliminating GBM stem cells and eradicating GBM.

本项目以研究恶性胶质瘤(GBM)干细胞外泌体介导的细胞通讯是否具有维持GBM干细胞动态平衡的作用及其作用机制为目的，采用无血清加细胞因子培养富集GBM干细胞，分离纯化GBM干细胞外泌体，用外泌体处理体非干性GBM细胞和体内接种细胞GBM细胞的荷瘤动物，用细胞生物学、分子生物学等方法，体内、外实验 相结合，研究GBM干细胞和非干性GBM细胞释放的外泌体数量和所含成分的差异，GBM干细胞外泌体对非干性GBM细胞干性表型、体内成瘤及干细胞比例的影响，阐明外泌体介导的细胞通讯在维持恶性胶质瘤干细胞动态平衡中的作用，揭示维持GBM干细胞的动态平衡的因素和机制；为研发同时靶向肿瘤干细胞和分化成熟肿瘤细胞的抗癌药物，或阻断肿瘤干细胞释放外泌体，或用外泌体负载靶向药物，阻断非干性肿瘤细胞去分化，转化为肿瘤干细胞，打破其动态平衡，从而彻底清除肿瘤干细胞，根治恶性胶质瘤措施的研究奠定理论基础和提供科学依据。

胶质母细胞干细胞(Glioblastoma stem cells, GSCs)是恶性胶质瘤治疗后肿瘤复发、进展及治疗耐受的罪魁祸首。胶质瘤干细胞维持于高度动态平衡中，其干性表型受特定肿瘤微环境因素影响，具有可塑性。分化成熟的非干性胶质瘤细胞具有去分化转化胶质瘤干细胞的潜能。外泌体是直径为 30-100nm的胞外囊泡，其内含有大量的生物活性物质，通过转运其活性物质，在细胞通讯中起重要作用。但胶质瘤干细胞释放的外泌体是否与分化成熟的非干性胶质瘤细胞去分化转化为胶质瘤干细胞有关尚未知晓。为本项目用无血清加细胞因子和添加剂悬浮培养、富集GBM干细胞，用PEG沉淀，联合试剂盒(exoEasy Maxi Kit)应用、分离、纯化、鉴定了GBM干细胞释放的外泌体，用GSC外泌体处理体外贴壁培养的非干性胶质瘤细胞(U251、U87)；用shNotch1慢病毒沉默Notch1，Notch信号抑制剂抑制Notch1信号通路。研究结果发现，GBM干细胞高表达干性及Notch信号通路相关蛋白；GBM干细胞外泌体富含Notch1蛋白，非干性胶质瘤细胞(U251、U87)外泌体含量低微；用GBM干细胞外泌体处理非干性胶质瘤细胞(U251、U87)，外泌体被摄入胞内；非干性胶质瘤细胞(U251、U87)的增殖活性、Neurosphere 形成、侵袭性及体内成瘤潜能显著增强；GBM干细胞外泌体处理过的胶质瘤细胞及所成瘤的组织中Notch信号通路相关蛋白及干性相关蛋白表达显著增加；Notch1干扰后的GBM干细胞及外泌体内Notch1蛋白表达显著降低，GBM干细胞Neurosphere 形成力降低；Notch1RNA沉默和Notch通路抑制剂处理后，GBM干细胞外泌体处理过的胶质瘤细胞内Notch信号通路相关蛋白及干性相关蛋白显著降低。恶性胶质瘤干细胞外泌体作为信息载体传递Notch1蛋白，激活Notch1信号通路介导了非干性胶质瘤细胞重编程或去分化，使之成为胶质瘤干细胞，增强其干性和成瘤性。靶向胶质瘤干细胞外泌体及Notch1信号通路，阻断胶质瘤干细胞外泌体释放或摄入从而杀灭胶质瘤干细胞可能是根治恶性胶质瘤的新策略，值得进一步深入研究。