叶酸修饰的牛奶外泌体携带siRNA-Notch1靶向胶质瘤干细胞治疗的作用机制研究

The presence of glioma stem cells（GSCs）is vital for the therapeutic effect of treatment to glioma. The stemness phenotype of GSCs is closely associated with relapse of glioma. Currently, the therapeutic strategy of targeting GSCs has been the hotspot of research. The latest study demonstrated that exosome, as a carrier of gene therapy, brings significant improvement to cancer treatment. Our previous study showed that Notch1 might be a target of stemness phenotypic regulation of GSCs, which indicated that siRNA-Notch1 carried by exosomes could be effective on glioma therapy. Moreover, we also found that folate modified milk derived exosomes are easier to be specifically absorbed by GSCs and could penetrate the model of blood-brain barrier. Based on these, we speculate that siRNA-Notch1 carried by folate modified milk derived exosomes may be a new strategy to targeted therapy of GSCs. To test this hypothesis, based on our preliminary results, this project will utilize technique of cellular and molecular biology to further elucidate the molecular mechanism of Notch1 regulating stemness phenotype of GSCs, and to explore the possibility of targeting GSCs with siRNA-Notch1 carried by folate-mediated milk exosomes. This project will provide new strategy, point out future translational direction and lay strong foundation for effective anti-glioma therapy.

胶质瘤干细胞的存在对胶质瘤治疗疗效的影响至关重要，其干性表型与胶质瘤复发密切相关，目前靶向胶质瘤干细胞的治疗策略已成为研究热点。近期研究表明，利用外泌体作为基因治疗载体，抗瘤疗效显著。我们的前期研究显示，Notch1可能是调控胶质瘤干细胞干性表型的重要靶点，提示利用外泌体携带siRNA-Notch1可能对胶质瘤治疗有效；此外，我们亦发现，叶酸修饰的牛奶外泌体主动靶向胶质瘤干细胞摄取并能穿透血脑屏障模型。基于此，我们推测，利用叶酸修饰的牛奶外泌体携带siRNA-Notch1，可能是靶向胶质瘤干细胞治疗的新策略。为验证该假设，本项目拟在已有工作基础上，采用细胞、分子生物学等技术，进一步阐释Notch1调控胶质瘤干细胞干性表型的分子机制，探索叶酸修饰的牛奶外泌体携带siRNA-Notch1靶向胶质瘤干细胞治疗的可行性，为有效的抗胶质瘤治疗新策略及可能的转化医学提供方向并奠定扎实的理论基础。

胶质瘤是最常见的原发性中枢神经系统肿瘤，预后差，复发率高。近来，越来越多的研究发现，胶质瘤干细胞的存在是抗胶质瘤治疗后肿瘤复发的关键，开展针对胶质瘤干细胞的靶向治疗至关重要。已有研究显示，肿瘤干细胞的干性表型维持与干性基因高表达密切相关，基于此，探寻胶质瘤干细胞干性表型关键调控基因并予以靶向干预，可能是抗胶质瘤治疗的新策略。然而，胶质瘤是颅内肿瘤，大多数抗肿瘤药物无法透过血脑屏障，因此，如何有效递送治疗性基因，进行精准的抗胶质瘤治疗是关键。牛奶外泌体作为药物/基因递送载体，具有其他细胞来源外泌体的特点，且更具有优势如下：较好的稳定性及生物相容性，低免疫原性，可透过血脑屏障，产量大，且价廉易得。但如何实现牛奶外泌体的靶向递送，仍然是课题需要进一步探索的问题。我们的前期研究发现，胶质瘤干细胞高表达叶酸受体α，而胶质瘤细胞株及正常人脑胶质细胞低表达叶酸受体α；提示应用叶酸修饰牛奶外泌体靶向胶质瘤干细胞递送干预胶质瘤干细胞干性表型维持之关键基因siRNA，可能是抗胶质瘤治疗的新策略。在本研究中，我们取得以下4点重要研究进展：1） Notch1在胶质瘤干细胞中高表达，且是调控胶质瘤恶性表型的重要分子之一，因此是通过基因治疗手段对胶质瘤进行综合治疗的可探索靶基因；2）叶酸是可用于修饰牛奶外泌体的靶向性小分子且通过叶酸修饰后胶质瘤干细胞样细胞对牛奶外泌体的摄取明显增加；3）叶酸修饰并荷载Notch1-siRNA的牛奶外泌体（FA-mEVs-Notch1-siRNA）体系具有体内外抗肿瘤疗效；4）沉默Notch1表达通过激活TNFR1-TRAF2信号调控胶质瘤干细胞样细胞的生长。以上研究结果提示，叶酸修饰并荷载Notch1-siRNA的牛奶外泌体体系可靶向治疗胶质瘤，是具有临床转化前景的治疗策略之一。此外，通过基础研究人员和市场的合作逐步建立和推进治疗性牛奶外泌体生产平台的标准化是未来努力的方向，越来越多的关注和资金投入必将加速治疗性牛奶外泌体的基础研究进展和抗肿瘤临床研究转化。