调控Wnt信号通路逆转脑胶质瘤干细胞耐药的作用机制研究

Glioma stem cells (GSCs), as "seed cells", are believed to be main causes of gliomas drug resistance and recurrence. Wnt pathway is critical in self-renewal and differentiation of tumor stem cells, and its abnormal activation leads to drug resistance of tumor cells. Our previous results indicated that GSCs cultured in serum free medium are resistant to cisplatin, doxorubicin and other chemotherapeutic agents. Moreover, Wnt signal in these cells activates. All these evidences imply us that intervention of Wnt signaling in GSCs probably serves as a new strategy to reverse drug resistance of gliomas. However, the role of Wnt pathway in GSCs drug resistance has not been reported at home and abroad. This project aims to optimize the technology of GSCs separation and culture, and clarify the Wnt pathway regulatory mechanism on drug resistance related genes and proteins of GSCs. And then we will evaluate the GSCs sensitivity to chemotherapeutic drugs after treatment with Wnt inhibitor in TMZ-induced drug-resistant cell model of glioblastoma cells. Finally, we further test the suspection in nude xenograft and providing experimental data for the application of the specific Wnt signaling inhibitors in glioblastoma clinical treatment.

脑胶质瘤干细胞(GSCs)作为脑胶质瘤的"种子细胞"，是脑胶质瘤耐药以及复发的根源。Wnt信号通路是调控肿瘤干细胞自我更新和分化的关键信号途径，该通路的激活与肿瘤细胞耐药密切相关。前期研究工作发现体外培养的GSCs对顺铂、阿霉素等多种化疗药物耐药，且在这些GSCs中Wnt信号通路异常激活，提示我们Wnt信号通路很可能在GSCs耐药中起关键调控作用。因此研究Wnt信号通路调控GSCs耐药的作用机制，将为逆转肿瘤细胞耐药提供新的思路。关于Wnt信号通路调控GSCs耐药机制研究国内外未见报道。本课题旨在优化体外分离培养GSCs技术基础上，研究Wnt信号通路对GSCs耐药基因及蛋白的调控机制；建立脑胶质瘤耐药细胞模型，确认抑制该通路能够提高GSCs对化疗药物的敏感性；最后，利用裸鼠移植瘤模型，在体内验证抑制Wnt通路能够逆转GSCs耐药，为Wnt信号通路抑制剂在脑胶质瘤治疗中的应用提供实验依据。

β-catenin是一种广泛参与细胞功能的细胞因子，本研究通过体内体外研究实验探索了β-catenin信号通路在胶质瘤干耐药及诱导分化的作用机制。结果发现：（1）在胶质瘤耐药细胞模型和胶质瘤干细胞模型中，β-catenin都明显上调，且与耐药状态明显相关；（2）耐药细胞中moesin/β-catenin信号通路明显激活，通过调控该信号通路可以明显逆转耐药，在采用针对这一信号通路设计的小分子化合物Daid002后，耐药细胞的增殖明显被抑制；（3）在胶质瘤肿瘤去分化模型中，moesin-β-catenin信号通路明显激活，Daid002同样能够显著抑制该信号通路，诱导胶质瘤细胞分化，体内体外发挥抗胶质瘤作用；（4）基于诱导分化的治疗策略，我们发现汉黄芩素通过抑制β-catenin信号通路促进胶质瘤分化，明显抑制胶质瘤的增殖。这些研究结果提示，Wnt/β-catenin信号通路在胶质瘤细胞耐药和诱导分化中都起着关键调控作用，其上游的moesin可以作为药物治疗胶质瘤耐药的新靶点。