MG53对脑缺血再灌注损伤的保护作用及其机制研究

发布时间：2018-08-12 14:10

【摘要】：研究背景:缺血所引的组织损伤是致死性疾病的主要原因,诸如脑卒中、冠状动脉硬化导致的心肌梗死等。临床发现,在组织器官缺血一定时间恢复血液供应后,却出现了更加严重的机能障碍,称之为缺血再灌注损伤(ischemia reperfusion injury,IRI)。在创伤性休克、外科手术、器官移植、烧伤、冻伤和血栓等血液循环障碍时,都会出现缺血后再灌注损伤。卒中已成为我国人口的第一位致残和死亡原因,且发病率有逐年增高的趋势,缺血性卒中是最常见的卒中类型,约占70%,病死率约为10%,致残率可达50%以上。如何降低脑IR对脑组织的损伤是防治重点。近年来,Mitsugumin 53(MG53)在细胞膜修复中的作用得到人们的重视,它的修复作用在骨骼肌细胞和心肌细胞中都得到了证实。MG53是三重结构域蛋白家族成员(Tripartite Motif Protein 72,TRIM72),属于肌特异性蛋白,在其氨基残端和羧基残端分别含有TRIM和SPRY结构域,主要分布在骨骼肌和心肌组织,其余组织很少表达或者没有表达。MG53蛋白分子与胞浆内囊泡和肌纤维膜紧密结合,参与细胞膜的修复,在细胞膜结构损伤后,MG53蛋白可在胞外钙的作用下由胞浆向胞膜转移,并在在膜破损处聚集,封闭破损的细胞膜,进而发挥其保护作用。脑IR的机制复杂,涉及自由基生成、细胞内钙超载、兴奋性氨基酸毒性、白细胞高度聚集和高能磷酸化合物缺乏等,针对这些机制,对脑IR的治疗采用钙拮抗剂、γ-氨基丁酸受体激动剂、AMPA受体拮抗剂、自由基清除剂等药物,但未能取得明显的效果。MG53可以直接针对细胞膜修复而发挥其保护作用,那么,作为MG53含量低或无MG53蛋白表达的组织器官(比如脑组织),给予外源性MG53是否具有保护作用呢?本课题拟采用SD大鼠脑IR模型进行研究,通过体内实验和体外实验相结合的方法共同验证MG53对脑缺血再灌注的保护作用。研究包括:1、MG53对神经元细胞的保护作用;2、rhMG53对SD大鼠脑缺血再灌注的保护作用;3、循环中MG53是否通过血脑屏障入脑发挥对脑缺血再灌注损伤的保护作用;4、rhMG53发挥对脑缺血再灌注的保护作用的信号通路。主要实验方法与实验步骤:1、验证脑组织及细胞是否存在mg53的表达我们采用野生型小鼠和mg53(-/-)基因敲除小鼠与sd大鼠的肌肉组织和脑组织,各种脑组织细胞系(小神经胶质细胞、星形胶质细胞、神经干细胞、神经细胞瘤细胞)作为研究对象,分别利用免疫印迹法和免疫组织化学法从蛋白水平和形态学方面验证mg53蛋白在脑组织及细胞上的表达与分布情况。2、mg53对脑组织和细胞在缺血再灌注和缺氧/复氧损伤的保护作用。我们采用sd大鼠和小鼠海马神经元细胞系ht22细胞,利用大脑中动脉堵塞法(middlecerebralarteryocclusion,mcao)建立sd大鼠局灶性脑组织缺血再灌注损伤模型和建立ht22细胞的缺氧/复氧模型,在体内体外实验,通过ttc染色、神经行为学评分、病理组织学和ldh水平检测等方法分析mg53对脑组织和细胞的保护作用。3、循环中mg53是否能通过血脑屏障入脑发挥对脑缺血再灌注损伤的保护作用。我们构建mg53转基因(tpa-mg53)小鼠,建立脑缺血再灌注模型后,利用免疫印迹法检测血液循环中mg53入脑后在脑组织中的表达,同时观察tpa-mg53小鼠脑缺血再灌注损伤后的保护作用。我们采用罗丹明染料标记rhmg53蛋白,静脉注射罗丹明标记的rhmg53和fitc-annexinv,分别利用小动物活体成像系统ivis和激光扫描共聚焦显微镜验证rhmg53通过血脑屏障进入脑组织发挥保护作用。4、rhmg53发挥脑损伤后的保护作用的信号通路。本部分实验利用sd大鼠建立脑缺血再灌注模型后,分为假手术组、假手术组+rhmg53组、脑缺血再灌注组、脑缺血再灌注组+rhmg53组,取脑提取脑组织蛋白,利用免疫印记法筛选再灌注损伤救援激酶(reperfusioninjurysalvagekinase,risk)通路和(或)生存活化因子增强(survivoractivatingfactorenhancement,safe)通路中关键信号分子的蛋白变化,明确rhmg53对脑组织中risk和(或)safe的调控作用,同时通过tunel染色和蛋白免疫印迹法观察脑组织凋亡程度的变化和凋亡蛋白caspase3的表达变化。在细胞实验部分,我们在神经干细胞中采用腺病毒转染gfp-mg53蛋白,使神经干细胞过表达mg53蛋白,并采用机械损伤的方法破坏细胞膜,观察mg53对细胞膜进行修复的作用。主要研究结果:1、脑组织及多种脑组织细胞中没有mg53蛋白的表达通过蛋白免疫印迹法和组织免疫荧光法明确了mg53蛋白在脑组织的多种细胞株中均没有表达。2、MG53对脑IR损伤和神经元细胞损伤均具有保护作用。2.1在动物体内实验中,通过TTC染色发现rh MG53可减少大鼠脑缺血再灌注损伤后梗死面积,病理学组织HE染色发现rhMG53能减轻脑组织的病理形态学改变,并对SD大鼠进行神经行为学评分,rh MG53能显著改善脑损伤后的神经体征。2.2在细胞体外实验中,在神经元细胞缺氧/复氧损伤后,rhMG53可以降低细胞损伤标志物LDH水平,提示外源性rh MG53对缺氧/复氧造成的神经元细胞损伤具有保护作用。3、循环中MG53可通过血脑屏障入脑发挥对脑缺血再灌注损伤的保护作用。t PA-MG53转基因小鼠循环中MG53和外源性rhMG53在脑缺血再灌注损伤后均可通过血脑屏障入脑,进而发挥其对脑损伤的保护作用。4、rhMG53发挥脑IR损伤保护作用的机制研究。4.1在细胞水平验证了MG53蛋白对神经干细胞的机械损伤具有膜修复作用:采用腺病毒转染GFP-MG53蛋白到神经干细胞中,使其细胞内MG53蛋白含量增加,发现MG53对神经干细胞的具有膜修复作用,且其修复神经细胞膜的作用依赖于氧化还原反应。4.2在动物水平验证了在脑缺血后再灌注早期静脉注射rh MG53蛋白能有效的激活RISK通路,增加Akt、GSK3β磷酸化水平,从而进一步抑制凋亡蛋白caspase 3的活性,抑制了再灌注损伤引起的组织细胞凋亡,发挥对脑组织的保护作用。结论:1、在脑组织及多种脑组织细胞中均没有MG53蛋白的表达。2、rh MG53可减少大鼠脑缺血再灌注损伤后梗死面积,并能显著改善脑损伤后的神经体征;同时,rh MG53可以发挥其对神经元细胞缺氧/复氧损伤的保护作用。3、循环中MG53和外源性rh MG53在脑缺血再灌注损伤后可通过血脑屏障入脑,到达损伤部位发挥其保护作用。4、MG53对神经干细胞具有膜修复作用,且其修复神经细胞膜的作用依赖于氧化还原反应;在脑缺血后再灌注早期后,rhMG53通过激活RISK通路提高Akt、GSK3β磷酸化水平,进而抑制Akt依赖的caspase 3的蛋白活性,抑制早期的脑组织细胞凋亡,,从而发挥对脑损伤的保护作用。
[Abstract]:BACKGROUND: Tissue injury induced by ischemia is the main cause of fatal diseases, such as stroke, myocardial infarction caused by coronary atherosclerosis and so on. Ischemic reperfusion injury occurs in traumatic shock, surgery, organ transplantation, burns, frostbite and thrombosis. Stroke has become the first cause of disability and death in China, and the incidence has been increasing year by year. Ischemic stroke is the most common type of stroke, accounting for about 70%, and the mortality rate is about 70%. In recent years, the role of Mitsugumin 53 (MG53) in the repair of cell membrane has attracted much attention. MG53 is a member of the triple domain protein family (Tripartite Motif P). Roein 72, TRIM72), a muscle-specific protein, contains TRIM and SPRY domains in its amino and carboxyl residues, mainly distributed in skeletal muscle and myocardial tissues, while few or no expression is found in other tissues. After injury, MG53 protein can transfer from the cytoplasm to the membrane under the action of extracellular calcium, and aggregate at the site of membrane breakage, seal the damaged membrane, and then play its protective role. For these mechanisms, calcium antagonists, gamma-aminobutyric acid receptor agonists, AMPA receptor antagonists, free radical scavengers and other drugs have been used in the treatment of brain IR, but no significant effect has been achieved. MG53 can directly target cell membrane repair and play its protective role, then, as a tissue organ with low or no MG53 protein expression (such as brain). Tissue), whether exogenous MG53 has protective effect? This study is to use the brain IR model of SD rats to study the protective effect of MG53 on cerebral ischemia-reperfusion by combining in vivo and in vitro experiments. The research includes: 1, the protective effect of MG53 on neurons; 2, the protective effect of rhMG53 on cerebral ischemia-reperfusion in SD rats. Protective effect of MG53 on cerebral ischemia-reperfusion injury through blood-brain barrier; 4. Signal pathway of rhMG53 on cerebral ischemia-reperfusion injury. Muscle and brain tissues of knockout mice and SD rats, and various brain tissue cell lines (microglia, astrocytes, neural stem cells, neurocytoma cells) were used as research objects. The expression of mg53 protein in brain tissues and fine tissues was verified by immunoblotting and immunohistochemistry, respectively. 2. The protective effects of mg53 on ischemia-reperfusion injury and hypoxia-reoxygenation injury of brain tissue and cells. We established focal cerebral ischemia-reperfusion injury in SD rats and mice by middle cerebral artery occlusion (mcao) using HT22 cells, a hippocampal neuronal cell line. The protective effects of mg53 on brain tissues and cells were analyzed by TTC staining, neurobehavioral score, pathological histology and LDH level. 3. Whether mg53 can enter the brain through blood-brain barrier during circulation can play a protective role in cerebral ischemia-reperfusion injury. We constructed mg53 transgenic mice and established cerebral ischemia-reperfusion model. The expression of mg53 in brain tissue was detected by Western blot. The protective effect of rhodamine dye-labeled rhmg53 protein was observed after cerebral ischemia-reperfusion injury in tpa-mg53 mice. Hmg53 and fitc-annexin V were used to validate the protective effect of rhmg53 through blood-brain barrier by IVIS and confocal laser scanning microscopy respectively. 4. rhmg53 played a protective role after brain injury. this part of the experiment used SD rats to establish cerebral ischemia-reperfusion model, divided into sham operation Group B, sham operation + rhmg 53, cerebral ischemia reperfusion group, cerebral ischemia reperfusion group + rhmg 53, brain tissue protein was extracted from the brain, and the key signals in the reperfusion injury rescue kinase (risk) pathway and (or) survival activating factor enhancement (safety) pathway were screened by immunoblotting. In the part of cell experiment, we used adenovirus to transfect gfp-mg53 protein into neural stem cells to make the nervous system more energetic. Mg53 protein was overexpressed by stem cells and damaged cell membrane by mechanical injury. the repairing effect of mg53 on cell membrane was observed. the main results were as follows: 1. there was no expression of mg53 protein in brain tissues and many kinds of brain tissues. the expression of mg53 protein in brain tissues was confirmed by immunoblotting and tissue immunofluorescence. No expression was found in the cell lines. 2. MG53 had protective effects on brain IR injury and neuronal cell injury. 2.1 In vivo, RH MG53 was found to reduce infarct size after cerebral ischemia-reperfusion injury in rats by TTC staining. HE staining of pathological tissues showed that rhMG53 could alleviate the pathological morphological changes of brain tissue, and it could be used in SD rats. Neurobehavioral score showed that rhMG53 could significantly improve the neurological signs after brain injury. 2.2 In vitro, rhMG53 could decrease the level of LDH after hypoxia/reoxygenation injury in neurons, suggesting that exogenous rhMG53 could protect neurons from hypoxia/reoxygenation injury. The protective effects of MG53 and exogenous rhMG53 on cerebral ischemia-reperfusion injury in t PA-MG53 transgenic mice were studied by blood-brain barrier. MG53 protein can repair the mechanical damage of neural stem cells. Adenovirus transfection of GFP-MG53 protein into neural stem cells increased the content of MG53 protein in the cells. It was found that MG53 had membrane repair effect on neural stem cells, and the effect of repairing neural cell membrane was dependent on redox reaction. At animal level, intravenous injection of RH MG53 protein can effectively activate RISK pathway, increase the phosphorylation levels of Akt and GSK3 beta, further inhibit the activity of apoptotic protein caspase 3, inhibit the apoptosis induced by reperfusion injury, and play a protective role in brain tissue. Conclusion: 1. There was no expression of MG53 protein in many kinds of brain tissues. 2. Rh MG53 could reduce the infarct size after cerebral ischemia-reperfusion injury in rats, and significantly improve the neurological signs after brain injury. At the same time, RH MG53 could play a protective role in hypoxia/reoxygenation injury of neurons. 3. MG53 and exogenous RH MG53 in circulation during cerebral ischemia-reperfusion injury. 4. MG53 has membrane repair effect on neural stem cells, and its effect on repairing neural cell membrane depends on redox reaction; rhMG53 increases Akt and GSK3 beta phosphorylation level by activating RISK pathway in the early stage of cerebral ischemia-reperfusion, and then inhibits Akt. Dependent caspase 3 proteins inhibit early apoptosis of brain cells and thus play a protective role in brain injury.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.3

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