【作者】 周帅；

【导师】 孙晓川；

【作者基本信息】 重庆医科大学 ， 外科学， 2013， 博士

【摘要】 载脂蛋白E（Apolipoprotein E,APOE表示其基因，apoE表示其编码蛋白）是中枢神经系统最主要的载脂蛋白，主要由星形胶质细胞产生。近年来，APOE基因多态性在脑损伤后继发性病理生理改变以及伤后转归中的作用越来越被更多的国内外学者所关注，普遍认为携带APOEε4等位基因的患者脑损伤易损性增加，并且可导致不良预后，但是相关机制目前仍不甚明了。缺氧是引起中枢神经系统细胞损伤最常见的原因，是脑创伤、缺血性卒中等疾病的基本病理过程。研究发现，星形胶质细胞缺氧后释放的多种细胞毒性介质对周围神经元可产生明显的损伤。越来越多的研究表明，星形胶质细胞结构和功能的改变在诸多疾病，如脑水肿、炎性脱髓鞘疾病、中枢神经系统退行性疾病等发病机制中扮演了重要的角色。星形胶质细胞是中枢神经系统中数量最多的细胞。然而目前对神经保护的研究主要集中于对神经元的保护，对其他非神经元细胞如星形胶质细胞的保护研究却少见报道。本课题以星形胶质细胞为研究对象，模拟脑损伤后脑组织缺血缺氧这一病理生理改变，探讨了apoE在此过程中的作用。本课题主要包括以下四方面的研究：一、星形胶质细胞的原代培养及缺氧损伤模型的建立采用酶消化法原代培养APOE基因敲除（APOE KO）鼠、APOE基因野生（APOE WT）鼠的星形胶质细胞、并纯化鉴定；参照相关文献，予细胞缺氧6h，构建星形胶质细胞缺氧损伤模型，透射电镜观察细胞超微结构形态变化。结果：光镜显示，细胞呈“铺路石”样排列，胞体较大呈不规则形，细胞免疫荧光发现，细胞能被抗GFAP的抗体识别；缺氧6h后，通过电镜均观察到细胞足突肿胀，线粒体形态不规则，空泡化，嵴不规则，可见细胞核凝集，染色质固缩、以及凋亡小体等凋亡细胞明显特征。结论：本部分实验成功构建了细胞的缺氧损伤模型，为后续实验奠定了基础。二、 apoE对星形胶质细胞缺氧性损伤的保护作用及机制研究1.将培养成熟的APOE KO及APOE WT星形胶质细胞均分为常氧组、缺氧组、常氧+T0901317组、缺氧+T0901317组，后两组细胞均为在常氧/缺氧前48h，向细胞培养基中加入促进apoE蛋白表达增加的肝X受体（liver X receptor，LXR）激动剂T0901317,并使其在培养基中的浓度为300nM。流式细胞仪分别检测以上各组细胞凋亡率情况。2.将培养成熟的APOE KO星形胶质细胞按照向其培养基中加入外源性apoE3的浓度分成6组：空白组、100nM组、300nM组、1000nM组、2000nM组、3000nM组，apoE3于缺氧前30min加入，予各组细胞缺氧6h后，流式细胞仪检测各组细胞凋亡率情况。3.将培养成熟的APOEKO及APOE WT星形胶质细胞均分为常氧组、缺氧组、常氧+T0901317组、缺氧+T0901317组，后两组细胞均为在常氧/缺氧前48h，向细胞培养基中T0901317,并使其在培养基中的浓度为300nM。缺氧6h后，收集各组细胞的细胞外液，通过氨基酸自动分析仪检测各组细胞外液中谷氨酸的水平。4.将培养成熟的APOE KO星形胶质细胞分为常氧组、缺氧组、缺氧+apoE3组、缺氧+MK801组、缺氧+apoE3+MK801组，后3组在缺氧前30min分别向培养基中加入人重组apoE3（1μM）、NMDA受体拮抗剂MK801（10μM）、apoE3（1μM）+MK801（10μM），流式细胞仪分别检测以上各组细胞凋亡率。5.将培养成熟的APOE KO及APOE WT星形胶质细胞均分为对照组、谷氨酸组、对照+T0901317组、谷氨酸+T0901317组，谷氨酸组为向培养基中加入谷氨酸（50mM），对照组即不作任何处理，保持原培养条件不变，后两组为在未加入/加入谷氨酸前48h，向培养基中加入LXR激动剂T0901317（300nM）；以上各组予继续培养24h；流式细胞仪分别检测以上各组细胞凋亡率。结果：1.缺氧6h后，APOE KO缺氧组细胞凋亡率明显高于APOE WT缺氧组(p<0.05)；T0901317能明显降低APOE WT缺氧组细胞凋亡率(p<0.05)，但是对APOE KO缺氧组无明显作用（p＞0.05）。2.从300nM组开始，细胞凋亡率较空白组明显降低(p<0.05)，1000nM组细胞凋亡率最低。3.缺氧后6h，APOE KO组、APOE WT组细胞外液中谷氨酸浓度较常氧组明显增加(p<0.05)，且APOE KO缺氧组明显高于APOEWT缺氧组(p<0.05)；T0901317能明显降低APOE WT缺氧组细胞外液中谷氨酸浓度(p<0.05)，但是对APOE KO缺氧组无明显作用（p＞0.05）。4.与常氧组相比，缺氧组、缺氧+apoE3组、缺氧+MK801组、缺氧+apoE3+MK801组细胞凋亡率均明显增加(p<0.05)，外源性加入apoE3或（和）MK801后，细胞凋亡率较单纯缺氧组明显降低(p<0.05)，但是apoE3、MK801对细胞缺氧性损伤的保护作用相似，且两者作用不叠加。5.谷氨酸处理24h后，APOE KO组、APOE WT组细胞凋亡率较对照组明显增加(p<0.05)，且APOE KO缺氧组明显高于APOE WT缺氧组(p<0.05)；T0901317能明显降低APOE WT谷氨酸组细胞凋亡率(p<0.05)，但是对APOE KO谷氨酸组无明显作用（p＞0.05）。结论：apoE对星形胶质细胞缺氧性损伤具有保护作用，且这种保护作用具有剂量-反应关系。apoE可抑制星形胶质细胞缺氧后NMDA受体的过度激活，降低谷氨酸的毒性，增加星形胶质细胞apoE的表达可能是缺氧性神经损伤保护的一种新途径。三、 apoE在星形胶质细胞缺氧性损伤中的表达变化及其机制将培养成熟的APOE WT鼠的星形胶质细胞分为常氧组、缺氧组和干预组（缺氧+ERK抑制剂），分别予缺氧组和干预组缺氧6h，干预组在缺氧之前向培养基中加入ERK信号通路抑制剂U0126， Westernblot法分别检测检测三组细胞apoE蛋白表达变化情况。结果：与常氧组和干预组相比，缺氧组星形胶质细胞表达的apoE蛋白明显增加(p<0.05)。结论：缺氧使星形胶质细胞apoE蛋白水平表达上调，ERK信号通路参与了该调节过程。四、 APOE基因多态性在星形胶质细胞缺氧性损伤过程中的作用1.原代培养APOE WT鼠、APOE转基因鼠（ε3、ε4）的星形胶质细胞，纯化鉴定，并构建6h细胞缺氧损伤模型；2.采用流式细胞仪分别检测各组细胞凋亡率及线粒体膜电位变化情况。结果：APOEε4组细胞早期凋亡率、线粒体膜电位下降程度明显高于APOEε3、APOE野生鼠组(p<0.05)。结论：缺氧损伤后早期，与APOEε3、APOE野生鼠组相比，APOEε4组星形胶质细胞线粒体膜电位下降程度更大，这可能导致更多的星形胶质细胞凋亡。更多还原

【Abstract】 In the brain, apolipoprotein E (APOE: gene; apoE: protein) ispredominantly synthesized by astrocytes. Several lines of evidence point toa direct role of apoE in modulating neuronal response to injury. Recently，mounting evidence have indicated that apoE genotype influences theoutcome from TBI. APOEε4is believed to be a negative factor that canresult in an unfavorable outcome from TBI，but the related mechanismsremain unclear.Hypoxia is the most common cause of cell damage in the brain. It canoccur during brain trauma, ischemic strokes. Astrocyte is the predominantcell type in the brain. Hypoxia-activated astrocytes can kill neighboringneurons through the realease of cytotoxic mediators and mounting evidencehas indicated that alterations in astrocyte played a crucial role in thepathogenesis of other disorders, such as brain edema, inflammatorydemyelinating diseases, neurodegenerative disorders and so on. While mostresearch is focused on the direct protection of neuronal cells, the role of non-neuronal cells, such as astrocytes that may be functional in thepathogenesis of these disorders, is relatively unclear. Our current study wasperformed from four aspects as follows.Part I Primary astrocyte cultivation and foundation of hypoxic injurymodelAstrocytes were separated from APOE wild type (WT) mice and APOEknockout (KO) mice and were determined by glial fibrillary acidic protein(GFAP) through immunofluorescence. Hypoxic injury models wereestablished by putting cells in the setting of hypoxia for6h. The changes ofcells and its mitochondria were observed by transmission electronmicroscope. Results: Cells can be recognized by GFAP-antibody. At6h ofischemia, swollen mitochondria were shown in the cytoplasm and cellswere shrunken and pyknotic; Apoptotic bodies were also observed.Conclusion: Astrocytic hypoxia injury model was successfully founded,providing foundation for next experiment.Part II Protective effects of apoE on astrocytes after hypoxic injury1. The APOE WT astrocytes and APOE KO astrocytes were dividedinto four groups: normoxia group, hypoxia group，normoxia+T0901317group and hypoxia+T0901317group. we added T090131748h prior tohypoxia and maintained in the culture media throughout the experiment.The apoptosis rate of astrocytes was determined6h later by flowcytometry(FCM).2. The APOE KO astrocytes were divided into six groups, among which, Control group,100nM group,300nM group,1000nMgroup,2000nM group,3000nM group were pretreated with full-lengthrecombinant human apoE30-3000nM30min before exposure to hypoxiarespectively. The apoptosis rate of astrocytes was determined6h later byFCM.3. The APOE WT astrocytes and APOE KO astrocytes were dividedinto four groups separately: normoxia group, hypoxia group，normoxia+T0901317group and hypoxia+T0901317group. we added T090131748hprior to hypoxia and maintained in the culture media throughout theexperiment. The culture of astrocytes exposed to hypoxia was collected.The concentration of glutamate in the culture was determined using theHitachi L-8800automatic amino acid analyzer.4. ApoE KO astrocyteswere divided into five groups: normoxia group, hypoxia group, hypoxia+apoE3group, hypoxia+MK801group and hypoxia+apoE3+MK801group, we pretreated apoE KO astrocytes with the recombinant humanapoE3(1μM) or the NMDA receptor antagonist, MK-801(10μM) or theboth,30min before hypoxia treatment. The apoptosis rate of astrocytes wasdetermined6h later by FCM.5. The APOE WT astrocytes and APOE KOastrocytes were divided into four groups separately: normal control (NC)group, glutamate group，NC+T0901317group and glutamate+T0901317group. we pretreated astrocytes with T0901317(300nM)48h prior toglutamate (50mM) treatment. The apoptosis rate of astrocytes weredetected by FCM. Results:1. The apoptosis rate in the WT hypoxia group was significantly lower than that in apoE KO group. No effect of T0901317was observed for hypoxia-treated apoE KO astrocytes.2. Pretreatment with300nM to3μM of apoE3resulted in partial protection compared to thecontrol group.3. The glutamate concentration in the culture of astrocyteswas increased significantly after being exposed to hypoxia for6h,especially in the apoE KO astrocytes. Under hypoxic condition,pretreatment with T0901317dramatically reduced the hypoxia-inducedincrease of extracelluar glutamate around WT astrocytes. However, thiseffect was not observed in apoE KO astrocytes.4. MK-801was sufficientto protect astrocytes from hypoxia-induced astrocyte apoptosis. Theprotective effect of MK801was similar to that observed in sister culturesexposed to hypoxia after pretreatment with1μM apoE3. Moreover, theeffects of MK-801and apoE3were not additive, that is, no furtherprotection was observed in cultures pretreated with both MK-801and apoEprior to hypoxia exposure.5. A significant excitotoxic response wasobserved in cultures exposed to50mM glutamate for24h both in WTastrocytes and apoE KO astrocytes, but the apoptosis rate in WT astrocyteswas significantly decreased compared with apoE KO astrocytes.Pretreatment with T0901317conferred partial protection againstglutamate-induced apoptosis in WT astrocytes. However, no T0901317protection was observed in apoE KO astrocyte cultures. Conclusion: apoEprotected astrocytes from hypoxia induced apoptosis in a dose-dependent manner. apoE exerts its protective effects both by reducing the level ofextracelluar glutamate around the astrocytes and decreasing the activationof NMDA receptors triggered by hypoxia. Approaches that elevate apoEsecretion in astrocytes might provide a novel strategy in the protection ofneuronal ischemic injury.Part III The changes of apoE expression in astrocytes exposed to hypoxicinjuryThe astrocytes of APOE WT mice were divided into normoxia group,hypoxia group and hypoxia+ERK inhibitor U0126group. The expressionof apoE of three groups cells were detected by Western blotting. Results:Compared to normoxia group and hypoxia+ERK inhibitor U0126group,the expression of apoE was highest in hypoxia group. Conclusion: Hypoxicinjury triggered the astrocytes to increasingly synthesize apoE, whichthrough the ERK signaling pathway.Part IV Effects of APOE polymorphisms on apoptosis of astrocytes afterhypoxic injuryAstrocytes were separated from APOE WT mice and APOE transgenicmice(ε3, ε4). Hypoxic injury models were established by putting cells inthe setting of hypoxia for6h. The apoptosis rate and mitochondrialmembrane potential were detected by flow cytometry. Results: Theapoptosis rate and decreasing degree of mitochondrial membrane potentialof ε4allele were significantly higher than the other two types(p<0.05). Conclusion: The higher of decreasing degree of mitochondrial membranepotential of APOEε4may induce more astrocytes apoptosis after hypoxia.更多还原