胶质瘤表达抗原2（GLEA2)通过ROS-JNK通路对神经胶质瘤杀伤作用的机制研究

As a common intracalvarium tumor, glioma is one of the worst prognostic tumors. Glioma expressed antigen 2（GLEA2） is a novel identified glioma antigen, GLEA2 was implied significantly correlated with prolonged survival in clinic studies. However, biological character and regulatory mechanism of GLEA2 in tumors are still not reported yet. Our premilinary study showed overexpression of GLEA2 in U251 glioma cells induced cell death. Moreover, reative oxidative species (ROS) was significantly increased in GLEA2-overexpressed U251 cells, subsquently, a key regulator in cell death pathway: c-Jun N-terminal kinase (JNK) was activated. Taken together, GLEA2 could be considered a regulator of glioma cell fate and a putative powerful tool against glioma growth. In light of our findings, we sight to investigate key role of GLEA2 in glioma development by generating glioma transplanted nude mice, and using various molecular techniques, such as tranfection, siRNA, fluorescence activated cell sorting (FACS) and immunohistochemistry. Furthermore, we will reveal molecular regulatory mechanism of GLEA2 mediated ROS generation and JNK activation by using multiple immunofluorescence,confocal microscope,western-blotting and so on. Finally, we will indicate contribution of ROS-JNK pathway to GLEA2 induced cell death. The study will offer a novel vision for evaluating prognosis and aid in further establishing a new target of gene therapy in glioma patients.

神经胶质瘤作为常见的颅内肿瘤，是全身预后最差的肿瘤之一。胶质瘤表达抗原GLEA2(Glioma-Expressed Antigen)是新发现的胶质瘤表达抗原，多项临床研究指出其和患者的预后息息相关。但是GLEA2在肿瘤中的生物学特性及调节机制在国际上尚未有文章报道。我们前期研究发现，GLEA2杀伤神经胶质瘤细胞，而且过表达GLEA2诱导细胞内活性氧(ROS)含量明显增多，进而激活了调节细胞死亡的关键性蛋白：JNK。提示GLEA2在胶质瘤发展中的负性调节作用。基于这一发现，本研究将利用基因转染，基因沉默技术，流式细胞术，免疫组化，免疫电镜技术，裸鼠肿瘤移植模型等方法深入探讨GLEA2对神经胶质瘤细胞的杀伤作用，通过多重免疫荧光，免疫印迹等方法研究GLEA2诱导ROS增高激活JNK杀伤肿瘤细胞的的分子机制。完成本课题对揭示胶质瘤发生发展，确立新的胶质瘤基因治疗靶点及患者的预后判断具有重要意义。

神经胶质瘤作为常见的颅内肿瘤，是全身预后最差的肿瘤之一。胶质瘤表达抗原GLEA2(Glioma-Expressed Antigen), 是新发现的胶质瘤表达抗原，又称为植物同源指蛋白PHF20 (plant homeodomain finger protein 20)。多项临床研究指出其和患者的预后息息相关。但是GLEA2在肿瘤中的生物学特性及调节机制在国际上尚未有文章报道。本研究主要分为三部分：1，研究GLEA2对神经胶质瘤细胞的杀伤作用，明确细胞死亡类型；2，研究了GLEA2对ROS激活MAPK-JNK通路诱导神经胶质瘤细胞死亡的作用；3，在体内，利用裸鼠肿瘤接种模型，验证GLEA2通过ROS-JNK对胶质瘤的杀伤作用及抗肿瘤活性。本研究利用了多种生物学技术，包括基因转染、基因沉默技术、流式细胞术、免疫组化、免疫印迹、免疫荧光、免疫电镜技术，裸鼠肿瘤移植模型等。研究发现GLEA2诱导PAR表达升高，并不诱导caspase3 和PARP的cleavage，而且caspase3抑制剂Z-VAD并不能抑制该死亡，采用双染色（Annexin IV和PI）流式细胞仪法和电镜形态学观察发现GLEA2诱导神经胶质瘤细胞坏死性细胞死亡。并且诱导细胞内和线粒体内ROS显著增高，降低线粒体膜电位，激活JNK。ROS抑制剂NAC和Catalase显著抑制GLEA2诱导的细胞死亡和JNK的活化，最后利用JNK抑制剂Sp，发现细胞存活率显著升高。在裸鼠背部注射纯化腺病毒后，发现与对照组相比，GLEA2组的肿瘤生长速度、大小和重量显著降低。完成本课题对深入了解GLEA2的分子生物学特性及其在胶质瘤发生发展中作用提供新思路，对确立新的胶质瘤基因治疗靶点及患者的预后判断具有重要意义。