胶质瘤细胞与星形胶质细胞的谷氨酸-谷氨酰胺代谢耦合-外科学(神外)专业论文.docxVIP

万方数据 万方数据 目 录 中文摘要·······················································3 英文摘要·······················································5 英文缩略表·····················································7 前言···························································8 第一部分 胶质瘤细胞株的建立及其部分特性的鉴定··················12 材料和方法·····················································12 结果···························································18 一、罕见胶质肉瘤细胞株(K308)的建立及特性鉴定······················18 二、具有干细胞特性的胶质瘤细胞株(YD309)鉴定························26 讨论···························································30 结论···························································32 第二部分 胶质瘤细胞与星形胶质细胞的谷氨酸-谷氨酰胺代谢耦合·····33 材料和方法·····················································34 结果···························································42 1. 胶质瘤细胞的谷氨酰胺摄取、谷氨酸分泌功能·······················44 2. 星形胶质细胞谷氨酸摄取及谷氨酰胺释放功能·······················50 3.共培养过程中胞外谷氨酸水平的限制·······························52 4. 胶质瘤细胞与星形胶质细胞对氧化应激的耐受能力····················55 5. 神经元与胶质瘤细胞、星形胶质细胞非直接接触共培养对神经元的影响····56 6. 胶质瘤细胞与星形胶质细胞共培养中诱导星形胶质细胞凋亡·············57 讨论···························································58 结论···························································66 参考文献·······················································67 综述···························································78 2 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、western blot 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