miR-106a/20b基因簇介导STAT3调控特异性致敏DC增强GSC.DC在靶向免疫治疗同源胶质瘤中的作用

It was known that glioma stem cells (GSC) are closely related to the occurrence and relapse of malignant glioma. This study will focus on targeting and eliminating GSC, based on our previous studies including the finished NSFC project titled as "Experimental study on glioma immunotherapy with the personal brain tumor stem cells (BTSC)" (No.81272439, to be checked and accepted in Dec.2013).The C57BL/6 mice and its homologous GL261 cell line will be used in the research to establish puromycin-resistant stable reporter cell lines carrying PNanog-GFP-T2A-Luc transgene. The antigens of glioma stem cells will be harvested by different ways including tumor lysate, apoptosis tumor antigen, fused cell antigen, total RNA extraction etc., for further sensitization of the homologous dendritic cells (DC). The optimal sensitization method will be determined by the following comparision and analysis of the function of mature DC and the activity of T lymphocytes(TLC). In the premise of the optimal sensitization, the STAT3 signal pathway of DC will be regulated by miR-106a/20b cluster in order to increase the effect of GSC+DC vaccine. The evalution of GSC or glioma cell reduction in the co-culture system of "GSC/glioma cell line and DC", along with all the DC functional indexes, and the measurement of IFN-γ secretion, tumor imagiology, morphology of focal tissue, plus the dynamic state of immunohestochemistry will be used in experiments in vitro and in vivo respectively, in order to justify the hypothesis about "Enhancing the effect of DC in immunotherapy of homologous glioma by regulating STAT3 pathway with miR-106a/20b cluster ".The aim of this research is to explore a new strategy of enhancing the efficacy of targeting immunotherapy on glioma with GSC, which will provide experimental data for personal therapy and anti-recidivation of glioma in clinical practice.

胶质瘤干细胞（GSC）与恶性胶质瘤发生发展及复发密切相关。为在前期工作（2013结题国基金项目"应用个体化BTSC靶向免疫治疗同源胶质瘤"）基础上，最大程度靶向消减GSC，本项目拟从增进DC参与靶向免疫治疗功能角度、继续深化靶向GSC的胶质瘤免疫治疗研究。拟以C57BL/6小鼠与同源胶质瘤细胞系GL261为客体，获取稳定表达Nanog的胶质瘤干细胞株，以裂解、凋亡、融合及全RNA提取法等不同方式获取GSC抗原并分别致敏同源DC，检测DC成熟功能状态，筛选GSC抗原对DC的最佳致敏方式；再于该最佳致敏条件下，以DC的STAT3为调控点、经miR-106a/20b基因簇调控、以GSC/GC削减程度为主要评估标准，体内外分组实验论证“miR-106a/20b基因簇通过调控由GSC特异性致敏DC的STAT3而增强GSC+DC靶向免疫治疗同源胶质瘤疗效”的假说。

脑肿瘤干细胞（BTSC）是一群具有干细胞特性的脑肿瘤细胞，与脑肿瘤的发生发展及复发密切相关，胶质瘤干细胞（GSC）则是 BTSC 的典型代表。目前有大量关于BTSC的理论研究，但是针对BTSC，特别是GSC的免疫治疗机制有待深入系统地探讨。本项目主要针对增进树突状细胞（DC）参与靶向免疫治疗功能角度、最大程度削减GSC方面进行研究，重点从构建、获取稳定胶质瘤干细胞株，以裂解、凋亡及全RNA提取法等方式制备GSC抗原并分别致敏同源DC，筛选GSC全抗原对DC的最佳致敏方式；再于该最佳致敏条件下，以miR-106a/20b基因簇为调控点、将STAT3作为下游通路分子，研究miR-106a/20b基因簇是否可以通过调控由GSC特异性致敏DC的STAT3分子而增强"GSC+DC"靶向免疫治疗同源胶质瘤疗效，进行了相关的实验探讨。研究发现：.（1）体外实验以裂解、凋亡及全RNA提取法等不同方式获取GSC抗原并分别致敏同源DC，综合筛选出最优方式为凋亡抗原致敏法；利用凋亡细胞致敏法制备胶质瘤细胞（GC）/GSC+DC瘤苗，以纯化的GSC为靶时，GSC+DC瘤苗杀伤效果显著优于GC+DC瘤苗，提示GSC+DC瘤苗对GSC有针对性杀伤的作用。.（2）miR-106a/20b的激活可能是增强GSC介导的DC抗肿瘤免疫应答的重要分子机制之一，通过下调DC STAT3的表达来削弱该通路的免疫负调作用。STAT3是DC成熟的重要调控因子，本研究通过生物信息学预测了STAT3 mRNA 的3'-UTR与miR-106a/20b之间的相互作用结合位点，并用双荧光素酶试验进行了验证。体外实验发现GSC+DC中STAT3水平的下调增加了CD8+ T淋巴细胞的生成，减少了Foxp3+ Treg细胞的生成,促使IFN-γ的分泌明显增加。进一步的研究发现，转染miR-106a/20b-mimics后的DC在体外可促进T淋巴细胞对GSC增殖的抑制，在实验小鼠体内可抑制同源胶质瘤的生长。.（3）课题的相关研究发现，焦亡关键分子GSDME的存在可以介导GC发生焦亡从而有效增强自然杀伤（NK）细胞对GC的杀伤能力，为NK细胞的抗胶质瘤作用及其机制提供了相关实验数据支持。成熟骨髓源性DC表达CD155，干扰GC+DC瘤苗CD155分子的表达可有效促进T淋巴细胞的增殖激活和分泌功能、改善体外GC特异性免疫杀伤效