胶质瘤细胞与星形胶质细胞的谷氨酸—谷氨酰胺代谢耦合

发布时间：2018-05-27 20:19

本文选题：胶质瘤 + 星形胶质细胞　；参考：《福建医科大学》2014年博士论文

【摘要】：目的研究显示胶质瘤细胞可通过释放大量的谷氨酸来促进自身生长、杀伤神经元，而星形胶质细胞是处理谷氨酸的主要载体，通过研究星形胶质细胞与胶质瘤细胞之间的相互作用，探讨星形胶质细胞在限制胶质瘤细胞所释放的胞外谷氨酸水平的作用，揭示内源性抑制胶质瘤生长及保护神经元的机制。为了尽量避免因现有的细胞株传代久远因素引起的实验结果的局限性，，本研究将建立胶质瘤细胞株，并利用传代10代以内的胶质瘤细胞进行实验。方法利用细胞培养技术、细胞示踪及凋亡检测、免疫细胞染色技术、westernblot技术、药物干预、高效液相色谱检测谷氨酸和谷氨酰胺、离子成像等技术，模拟体内胶质瘤生长过程，验证如下假说：1、在胶质瘤生长的早期，其周围包绕大量正常的星形胶质细胞，此时（1）胶质瘤细胞是否高表达谷氨酰胺酶，而不表达或错位表达与谷氨酰胺酶作用相反的谷氨酰胺合成酶；（2）胶质瘤细胞能否大量摄取谷氨酰胺进入胞内并转化为谷氨酸分泌到细胞外；（3）星形胶质细胞能否有效地摄取胶质瘤细胞所分泌的谷氨酸；2、在胶质瘤生长的后期，其数量上超过周围包绕的星形胶质细胞，此状态下谷氨酸-谷氨酰胺代谢耦合是否被打破：（1）胶质瘤细胞所释放的氨及氧化应激能否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，引起神经元的损伤；（2）大量的胶质瘤细胞能否通过氧化应激等造成星形胶质细胞的损伤甚至死亡。结果根据以下特征（1）胶质瘤细胞高表达谷氨酰胺酶，但谷氨酰胺合成酶错位表达。而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，再主要通过胱氨酸-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细胞所分泌的谷氨酸，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，此状态下谷氨酸-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显著抑制星形胶质细胞的谷氨酸摄取功能，而使共培养中的胞外谷氨酸水平明显升高；（2）胞外谷氨酸明显升高到能够诱导神经元的损伤及死亡；（3）胶质瘤细胞所产生的大量氨和氧化应激还能明显诱导星形胶质细胞凋亡。结论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胶质瘤的谷氨酸释放所主导，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，使胶质瘤进入快速生长阶段。因此，保持星形胶质细胞与胶质瘤细胞之间谷氨酸-谷氨酰胺代谢耦合的平衡以及维持星形胶质细胞的主导作用，可能可以作为内源性抗肿瘤生长和保护神经元的机制。
[Abstract]:Objective to show that glioma cells can promote their growth and kill neurons by releasing a large amount of glutamic acid, and astrocytes are the main carrier of glutamate treatment. By studying the interaction between astrocytes and glioma cells, astrocytes are used to explore the extracellular valley of astrocytes in the restriction of glioma cells. In order to avoid the limitations of the experimental results caused by the long distant factors of the existing cell lines, this study will establish glioma cell lines and make use of glioma cells within 10 generations to carry out experiments.
Methods using cell culture technique, cell tracer and apoptosis detection, immuno cell staining technique, Westernblot technology, drug intervention, high performance liquid chromatography to detect glutamic acid and glutamine, ion imaging techniques to simulate the growth process of glioma in vivo, verify the following hypothesis: 1, around the early growth of glioma, the surrounding package around a large number of normal. Astrocytes, at this time (1) whether glioma cells are highly expressed as glutaminase, and do not express or misrepresent glutamine synthetase opposite to glutaminase; (2) whether glioma cells can absorb glutamine into the cell and convert into glutamic acid out of the cell; (3) whether astrocytes are effective or not. Uptake of glutamate secreted by glioma cells; 2, in the late stage of the growth of glioma, the number exceeds the surrounding astrocytes. In this state, whether the glutamic glutamine metabolic coupling is broken: (1) whether the ammonia and oxidative stress released by glioma cells can inhibit glutamic acid to a certain extent The level of extracellular glutamic acid increased significantly in co culture, causing neuronal damage, and (2) whether a large number of glioma cells could cause astrocyte damage or even death through oxidative stress.
Results according to the following characteristics (1) glioma cells expressed glutaminase high, but glutamine synthetase was misplaced, and astrocytes highly expressed glutamine synthetase; (2) glioma cells were able to absorb glutamine into cells and convert into glutamic acid, and then secreted mainly through cystine glutamic acid exchange protein. (3) in the process of co culture of astrocytes and glioma cells, astrocytes can effectively absorb glutamic acid secreted by glioma cells and prove that there is a metabolic coupling between glutamate and glutamine in astrocytes and glioma cells. In the later period of the growth of gelatoma, the volume of glioma increases and the number of glioma is increased. In addition, the metabolic coupling of glutamic glutamine was broken in this state: (1) the ammonia and oxidative stress released by glioma cells significantly inhibited the glutamate uptake of astrocytes and increased the level of extracellular glutamic acid in co culture; (2) extracellular glutamic acid significantly increased to the ability to induce neuronal damage and death. (3) a large amount of ammonia and oxidative stress produced by glioma cells can also significantly induce astrocyte apoptosis.
Conclusion in the early stage of glioma growth, glioma cells and astrocytes are coupled with glutamic glutamine metabolism, and with the growth of glioma, the metabolic coupling of glutamic acid is gradually dominated by the release of glutamic acid in glioma, causing damage and death of neurons and astrocytes, and further to glioma. Growth and invasion provide space, while extracellular elevated glutamic acid may further promote the growth of glioma cells and make glioma into the rapid growth stage. Therefore, it is possible to maintain the balance of glutamate metabolic coupling between astrocytes and glioma cells and the dominant role of maintaining astrocytes in astrocytes. As an endogenous mechanism of anti-tumor growth and protection of neurons.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.4

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