恶性人脑胶质瘤辐射抗拒相关新基因c-Fos的功能验证及机制研究

The hypothesis is based on previous project of international cooperation program which was completed in the United States by the applicant: we established orthotopic glioma mouse model by injecting fresh surgical specimens of human gliomas into mouse brain, and then simulated classic fractionated radiation in the treatment group. Global gene expression profiles of control group, radiation group and relapsed group were then analyzed using microarrays. c-Fos was identified and verified to be upregulated in radiation and relapsed groups mouse models . In this study, we plan to downregulate or overexpress the c-Fos gene by lenti virus in glioma cell lines and glioma stem cells, then analyze the radiosensitivity and DNA damage repair capacity in them, cell cycle distribution and apoptosis were detected as well; Then explore the impact of c-Fos expression on the ability of invasion by transwell assay and investigate the changes of DNA damage repair and cell cycle distribution pathways by Western-blot;Figure out the possible molecular mechanisms by gene array;At last, verify its influence on radiosensitivity and analyze its prognostic value in glioma patients by immunostaining 241 cases of clinical samples. In this study, we would like to clarify the role of c-Fos gene in glioma radiosensitivity and the involved molecular mechanisms, and provide novel target for radiosensitization of glioma treatment in near future.

申请者前期在美国完成的国际合作课题中通过直接注射新鲜胶质瘤手术标本建立人脑胶质瘤小鼠脑原位模型，全基因组芯片分析多例分次放射治疗前后及治疗失败复发组脑原位胶质瘤标本，筛选并验证共同显著差异基因c-Fos。本研究在上述研究基础上，将在胶质瘤细胞系，胶质瘤干细胞及原位动物模型水平分析慢病毒下调或过表达c-Fos基因后对放射敏感性的影响，检测DNA损伤修复能力、细胞周期分布和凋亡等阐述c-Fos介导胶质瘤辐射抗拒的机制，进一步细胞侵袭实验探讨其联合放射线对侵袭力的影响，蛋白质印迹研究DNA损伤修复和细胞周期调节通路的改变；通过基因芯片筛选下调c-Fos后的分子通路改变，探索可能的调控关系；在有完整预后资料的241例胶质瘤临床标本中验证c-Fos蛋白与放射敏感性及预后的关系。本研究将初步阐明c-Fos对胶质瘤放射敏感性的影响及分子机制，为以其作为放射增敏靶点的脑胶质瘤治疗探索新策略。

申请者通过直接注射新鲜胶质瘤手术标本建立人脑胶质瘤小鼠脑原位模型，全基因组芯片分析多例分次放射治疗前后及治疗失败复发组脑原位胶质瘤标本，筛选并验证共同显著差异基因c-Fos。本研究在上述研究基础上，为进一步研究c-Fos基因对于胶质瘤放射敏感性的影响，和探索其分子机制，在胶质瘤细胞系使用慢病毒下调c-Fos基因后，进一步分析其对放射敏感性的影响，检测DNA损伤修复能力、细胞周期分布和凋亡等。在胶质瘤细胞系中，下调c-Fos联合放射线能显著减少胶质瘤细胞集落的形成，突破G1/S期阻滞，诱导G2M期增多。联合治疗能增强DNA损伤，并且延迟DNA损伤修复，促进凋亡。在有完整预后资料的近80例胶质瘤临床标本中验证c-Fos蛋白与放射敏感性及预后的关系。本研究将初步阐明c-Fos对胶质瘤放射敏感性的影响及分子机制，为以其作为放射增敏靶点的脑胶质瘤治疗探索新策略。