胶质瘤中IDH1突变的肿瘤生长抑制与放化疗增敏作用及其机制

Isocitrate dehydrogenase 1 gene (IDH1) mutation which is characterized by glioma occurs frequently and closely relates to a better outcome. However, the protective mechanism and value of treatment in glioma still remain indefinite. Our previous studies found that the level of intracellular reactive oxygen species (ROS) elevated while the growth of glioma cells, which overpressed mutated IDH1 gene , was significantly inhibited. We speculate that the rise in ROS level induced by IDH1 mutation is an important mechanism of tumor growth inhibition, and may have sensitisation effect to radiotherapy and chemotherapy. On the basis of previous studies, the project will use the technologies of gene overexpression, siRNA interference, protein chip and tumor-bearing animal models to further verify the glioma growth inhibition effect of IDH1 mutation, to investigate the potential ROS-mediated signaling pathway, to make clear the radiotherapy and chemotherapy sensitisation effect stimulated by IDH1 mutation and screen sensitive chemotherapy drugs through in vitro and in vivo experiments. The purpose of the project is to elucidate the specific mechanisms of the improvement in prognosis of IDH1 mutation, to provide the experimental basis for predicting the therapeutic effect and selecting individualized treatment scheme of IDH1 mutated glioma, and to afford novel targets for targeted therapy of glioma.

异柠檬酸脱氢酶1(IDH1)基因突变在脑胶质瘤中发生频繁，具有胶质瘤特异性，与患者预后改善密切相关，但其保护机制和对肿瘤治疗的意义尚不明确。前期研究发现：胶质瘤细胞中过表达突变型IDH1后，胞内活性氧(ROS)水平升高，肿瘤细胞生长明显抑制。我们推测，IDH1突变体诱导ROS水平升高是抑制肿瘤生长的重要机制，且可能具有放化疗增敏作用。本项目在前期研究基础上，利用基因过表达、siRNA干扰、蛋白质芯片、荷瘤动物模型等技术进一步验证IDH1突变的肿瘤生长抑制作用，探讨ROS介导的可能信号通路；通过体外、体内实验明确IDH1突变的胶质瘤放化疗增敏作用并筛选化疗敏感药物。通过本项目的研究，阐明IDH1突变改善预后的具体机制，为IDH1突变对胶质瘤治疗效果的预测和个体化治疗方案的选择提供实验基础，并为胶质瘤靶向性治疗提供新的靶标。

异柠檬酸脱氢酶1（IDH1）基因突变在胶质瘤中发生频繁，具有特异性，是一个独立的预测有利的预后因素。我们的前期实验发现，在胶质瘤细胞中转染IDH1-R132H突变体后，细胞周期进程受阻，细胞增殖下降、凋亡增加，导致肿瘤细胞生长抑制。同时发现，转染IDH1-R132H后，胶质瘤细胞内还原型辅酶Ⅱ（NADPH）、还原型谷胱甘肽（GSH）含量下降，活性氧（ROS）水平明显升高。本项目在前期工作基础上进一步的研究结果为：（1）在91例星形细胞胶质瘤中通过直接测序法检测IDH1基因突变分布，结果提示IDH1突变率与病理级别成负相关；生存分析与单因素分析提示IDH1突变型胶质瘤患者中位生存期较IDH1野生型患者明显延长。（2）采用PCR定点突变技术，获得IDH1-R132H的慢病毒质粒，转染胶质瘤细胞后基因表达谱显示，1255个基因上调和1862个基因下调，进一步功能分析显示IDH1突变主要影响细胞代谢过程，有丝分裂周期和凋亡。（3）IDH1-R132H转染U87胶质瘤细胞，采用免疫荧光染色观察细胞增殖，TUNEL法检测细胞凋亡，WST法绘制细胞生长曲线，并进一步采用比色法检测细胞内还原性烟酰胺腺嘌呤二核苷酸磷酸（NADPH）、还原性谷胱甘肽（GSH）含量，荧光探针检测细胞内活性氧（ROS）含量，结果显示：表达IDH1-R132H突变体的胶质瘤细胞生长明显抑制，细胞内GSH含量降低和ROS蓄积可能是其重要机制。（4）胶质瘤细胞中过表达IDH1-R132H突变体具有化疗增敏作用，明显增加了替莫唑胺（TMZ）和顺铂（CDDP）的化疗敏感性，GSH耗竭和ROS蓄积可能是IDH1突变化疗增敏作用的重要原因和机制。.通过本项目的研究，阐明了IDH1突变改善预后的具体机制，明确了ROS水平升高可能是IDH1突变抑制胶质瘤肿瘤细胞生长的具体分子机制，国内外未见类似报道，具有明确的原创性；进一步通过体外、体内实验证实了IDH1突变的胶质瘤化疗增敏作用，并筛选出化疗敏感药物为替莫唑胺和顺铂，具有重要的临床应用及推广价值。