胶质瘤细胞与星形胶质细胞的谷氨酸-谷氨酰胺代谢耦合及氧化应激损伤

A critical component of glioma pathology is the toxicity it brings to neighboring brain tissues. Our previous studies revealed that glioma cells release excitotoxic levels of glutamate (Ye and Sontheimer, Cancer Research 1999). Further studies suggest that glutamate can stimulate the growth of glioma cells. However, little is known about the function of normal astrocytes in the progression of glioma. In light of the pivotal role of astrocytic glutamate transport in maintaining glutamate homeostasis in the brain, we proposed that normal astrocytes are capable of limiting the neurotoxicity and the growth of glioma cells. However, such capability is vulnerable to aberrant glioma metabolism. Using ion imaging, HPLC analysis, immunohistochmistry and other related techniques, we will test the following hypotheses: (1) Normal astrocytes sequester glutamate released by glioma cells, rendering neuronal protection while reducing the proliferation-stimulating effects of glutamate. In glioma cells, glutamine is broken down into glutamate and ammonia, glutamate then either enters the TCA cycle or is released and mostly transported into neighboring astrocytes. Astrocytes synthesize glutamine from glutamate and predominantly release glutamine into extracellular space, the glutamine is then transported into glioma cells, instead of neurons, to repeat this glutamate-glutamine cycling.(2) Glutamate transport and glutamine synthesis increase ATP demand in astrocytes and the resulting upregulation of mitochondria activity increases the production of reactive oxygen species (ROS). Meanwhile, constitutive production of ammonia from the hydrolysis of glutamine by glioma significantly increases ammonia concentration in the vicinity of glioma, locally creating pathological conditions mimicking hepatic encephalopathy, in which astrocytes are most vulnerable to high ammonia levels, manifested by increase of ROS production and cell damage. (3) Astrocytes and glioma cells both require a normal transmembrane Na+ gradient for proper function and survival. However, the astrocytic Na+ gradient is much more vulnerable to oxidative stress than the glioma Na+ gradient; increased oxidative stress induced by glioma cell activities in and around the tumor can likely selectively compromise the astrocytic Na+ gradient,drastically reduce astrocytic glutamate uptake and compromise neuro-protection and tumor limiting effects accompanying astrocytic glutamate transport. Above all, this study may reveal endogenous glioma-limiting mechanism and means to preserve and utilize this mechanism in patient care.

研究显示胶质瘤细胞的谷氨酸释放有诱发癫痫、杀伤神经元及促进胶质瘤自身生长等作用，而星形胶质细胞的谷氨酸摄取是遏制细胞外谷氨酸水平过度升高的关键，但其对胶质瘤释放的谷氨酸的可能影响并不清楚。本项目的核心假设是"正常"星形胶质细胞通过谷氨酸摄取可抑制胶质瘤的生长与神经毒性，然而胶质瘤的代谢产物使谷氨酸摄取受损于氧化应激，从而减弱或丧失了对胶质瘤细胞的遏制作用。本研究拟结合细胞离子成像、HPLC代谢分析、胶质瘤标本免疫组化等技术，来论证下列假设：（1）星形胶质细胞与胶质瘤细胞间形成谷氨酸-谷氨酰胺的代谢、摄取与释放的耦合；（2）耦合中的能量负荷及胶质瘤谷氨酰胺代谢产生的氨对相邻星形胶质细胞形成氧化应激；（3）星形胶质细胞跨膜钠离子梯度对氧化应激的耐受力远低于胶质瘤细胞，钠离子梯度的丧失抑制了谷氨酸摄取。该研究可为了解、保护与利用内源性的抗胶质瘤机制打下理论基础。

胶质瘤细胞通过释放大量的谷氨酸促进自身生长、杀伤神经元、诱发癫痫的发生，而星形胶质细胞是处理谷氨酸的主要载体。根据以下特征（1）胶质瘤细胞高表达谷氨酰胺酶，而谷氨酰胺合成酶错位表达，但星形胶质细胞高表达谷氨酰胺合成酶；（2）胶质瘤细胞能够大量摄取谷氨酰胺进入细胞内并转化成为谷氨酸，主要通过胱氨酸-谷氨酸交换蛋白分泌到细胞外；（3）在星形胶质细胞与胶质瘤细胞共培养过程中，星形胶质细胞能够有效摄取胶质瘤所分泌的谷氨酸，证明星形胶质细胞与胶质瘤细胞之间存在谷氨酸-谷氨酰胺代谢耦合。而在胶质瘤生长的后期，胶质瘤体积增大，数量增多，其数量上远远超过周围包绕的肿瘤，利用共培养（星形胶质细胞数量远远小于胶质瘤细胞）手段模拟体内生长过程发现谷氨酸-谷氨酰胺代谢耦合被打破：（1）胶质瘤细胞所释放的氨及氧化应激在一定程度上抑制星形胶质细胞的谷氨酸摄取功能，而使共培养中胞外谷氨酸水平明显升高；（2）共培养体系中，在胶质瘤细胞大量存在情况下，胞外升高的谷氨酸明显升高能够诱导神经元的损伤及死亡；（3）胶质瘤细胞所产生的氨和氧化应激不仅能够损伤星形胶质细胞的谷氨酸摄取功能，在胶质瘤细胞大量存在的情况下能够明显诱导星形胶质细胞凋亡。基于上述模拟体内胶质瘤细胞生长的过程，提出在胶质瘤生长的早期，胶质瘤细胞与星形胶质细胞存在谷氨酸-谷氨酰胺代谢耦合，而随着胶质瘤的生长，谷氨酸-谷氨酰胺代谢耦合逐渐被打破，造成神经元和星形胶质细胞的损伤和死亡，进一步为胶质瘤的生长和侵袭提供空间，同时胞外升高的谷氨酸可能进一步促进胶质瘤细胞的生长，胶质瘤细胞进入快速生长阶段。因此，保持星形胶质细胞与胶质瘤细胞之间谷氨酸-谷氨酰胺代谢耦联的完整性可能可以作为内源性抗肿瘤生长和保护神经元的机制。