TIGAR调节IDH1突变型胶质瘤干细胞放射敏感性的机制研究

In recent years, it has been discovered that the radio-chemo-resistance of glioma stem cells (GSCs) is one of the most important reasons of tumor recurrence. Therefore, reversal of the radioresistance of GSCs becomes particularly critical to improve the clinical effect. Isocitrate dehydrogenase-1 (IDH1) is an important molecular bio-marker of glioma development, the mutation rate of IDH1 is as high as 50-80% in grade II-III gliomas and secondary malignant gliomas . The present project focused on the monotony of NADPH anabolism in IDH1 mutant GSCs. By inhibiting TIGAR expression and downregulating the flux of pentose phosphate pathway, the redox balance of IDH1 mutant GSCs was undermined thereby cells were radiosensitized. In this study, IDH1 mutant GSCs will be constructed by CRISPR/Cas9 system, and the transformation of metabolic pathway in IDH1 mutant GSCs will be examined. By investigating the antioxidants and redox signaling, the mechanisms of TIGAR regulated tumorigenicity and radiosensitivity of IDH1 mutant GSCs will be elucidated. Finally, GSCs-implanted orthotopic model will be used to further study the radiosensitizing approaches. In conclusion, the current project aims to provide a reliable and effective radiosensitizing therapeutic model for glioma clinical treatment.

胶质瘤干细胞(GSCs)的放化疗抵抗是胶质瘤治疗后复发的重要原因，逆转GSCs的辐射抗性被认为是提高胶质瘤疗效的关键。异柠檬酸脱氢酶-1(IDH1)突变是促进胶质瘤发生发展的重要分子标志，在WHO II-III级胶质瘤以及继发的恶性胶质瘤中IDH1突变率高达50-80%。本课题拟利用IDH1突变型GSCs中NADPH合成途径相对单一的代谢弱点，通过抑制TIGAR表达下调细胞磷酸戊糖途径的活性，从而破坏细胞氧化还原平衡，达到辐射增敏的效果。本课题拟通过CRISPR/Cas9技术构建IDH1突变型GSCs，鉴定IDH1突变型GSCs的代谢表型；检测抑制TIGAR表达前后细胞的氧化还原信号通路，阐释TIGAR调节IDH1突变型GSCs成瘤能力及放射敏感性的相关机制；通过人脑GSCs裸鼠原位荷瘤模型进一步验证抑制TIGAR表达的放疗增敏效果，从而提出一种有效的胶质瘤辐射增敏新模式。

异柠檬酸脱氢酶1（IDH1）是三羧酸循环的一个关键限速酶，临床研究显示IDH1突变普遍存在于WHOII级和III级胶质瘤以及继发性胶质母细胞瘤中，并且IDH1突变型患者比IDH1野生型患者对放化疗的反应更好，但其中的机制尚不明了。本研究通过构建IDH1 R132H突变胶质瘤细胞株研究IDH1 R132H突变对胶质瘤细胞放射敏感性的影响。细胞克隆实验检测胶质瘤细胞在0、2、4、6、8 Gy X射线照射后的克隆形成能力，结果显示各剂量点IDH1 R132H突变型组胶质瘤细胞的克隆存活分数显著低于对照组。细胞免疫荧光检测电离辐射后细胞核内γ-H2AX foci的数目变化，结果表明IDH1突变延缓了细胞DNA损伤修复的进程。X射线照射后4 h、12 h突变组细胞的γ-H2AX foci数目较对照组显著增加。细胞周期实验表明IDH1 R132H突变细胞能够延迟电离辐射后引起的周期阻滞。胶质瘤细胞经4 Gy X射线照射后12 h，IDH1 R132H组胶质瘤细胞的G2/M期细胞数是对照组细胞的2倍。Western blot 实验结果显示IDH1 R132H突变下调TIGAR蛋白的表达，而IDH1 R132H的特异性抑制剂AGI-5198处理后可逆转TIGAR下调。本项目研究揭示，电离辐射后IDH1 R132H突变型胶质瘤细胞较野生型细胞更为敏感，DNA损伤修复进程延缓，更多的细胞发生G2/M期细胞周期阻滞。在分子机制上，IDH1 R132H突变有可能是通过增加抑制TIGAR蛋白来调控胶质瘤细胞的放射敏感性的。..关键词：IDH1突变、胶质瘤、DNA损伤修复、放射敏感性