胞外Peroxiredoxin 2在蛛网膜下腔出血后早期脑损伤中的作用研究

发布时间：2018-05-20 20:05

本文选题：Peroxiredoxin + 2　；参考：《第二军医大学》2016年博士论文

【摘要】：背景:蛛网膜下腔出血(subarachnoid hemorrhage,SAH)是三大脑血管疾病之一,致死率、致残率高,危害极大。SAH后蛛网膜下腔中常常留有大量红细胞及其裂解产物,这些物质可触发一系列复杂的病理生理改变,包括脑水肿、氧化应激、炎性反应、细胞毒性作用等。而且,这些改变相互交错、一起作用,最终导致神经细胞损伤、神经功能障碍。Peroxiredoxin蛋白家族(peroxiredoxins,Prxs)是一类真核细胞中普遍存在的、起清除细胞内过氧化物作用的抗氧化剂,同时介导调控过氧化氢水平的信号通路,并参与许多疾病的发生发展。进一步研究发现,Prxs出胞后失去原有的细胞保护功能,所扮演的角色转变。例如,当脑缺血卒中发生后,Prxs释放至细胞外,启动严重的炎性反应,作用机制可能为通过Toll样受体2(toll-like receptor 2,TLR2)与Toll样受体4(toll-like receptor 4,TLR4)、激活核因子-κB(nuclear factor-κB,NF-κB)途径启动损伤相关分子模式(damage-associated molecular pattern,DAMP)型炎性反应,导致神经细胞损伤加剧,同时伴随Prxs的进一步释放,促炎作用级联放大,最终出现严重的损伤结果。胞外的Prxs表达水平也会相应升高。而Peroxiredoxin 2(Prx2)作为Prxs家族六个成员之一,在红细胞中蛋白含量居第三(仅次于血红蛋白),在神经元中也有广泛表达。由于Prx2这一独有的细胞分布特点,其在SAH后EBI中可能会扮演极其重要的作用。理由是:红细胞及其裂解产物是SAH后EBI的重要致伤因素,激活诸多损伤通路,而神经元恰恰是这些损伤通路最终的靶细胞,因果交错,作用叠加。因此,我们提出假设:当SAH发生后,蛛网膜下腔中红细胞会因衰老或肿胀等因素逐渐破裂、释放出Prx2;脑组织中神经元因缺血、炎性反应等因素出现损伤、坏死也会释放出Prx2,导致脑脊液、脑皮层间质中Prx2(即胞外Prx2)表达异常升高。-2-这些胞外prx2除了可能会直接导致神经细胞损伤外,还可能会启动、并介导damp型炎性反应,加剧神经元进一步损伤,并伴随prx2进一步释放,作用级联放大,最终在sah后早期脑损伤中扮演极其重要的角色。方法:本课题首先运用western-blotting技术观察prx2在红细胞中表达情况,再运用免疫荧光技术分别从体内、体外观察prx2在各种脑细胞中分布,验证脑组织中prx2是否仅在神经元上表达。随后,采用兔枕大池一次注血方法建立大动物实验性蛛网膜下腔出血模型,用western-blotting、酶联免疫吸附实验(enzyme-linkedimmunosorbentassay,elisa)技术检测红细胞裂解液组与红细胞悬液组、对照组中脑组织中prx2与脑脊液(cerebrospinalfluid,csf)中prx2、炎性因子(il-6、tnf-α)的含量,用免疫组织化学、nissl染色、he染色等技术观察早期脑损伤及基底动脉痉挛情况,初步得出胞外prx2可能参与早期脑损伤作用中。其次,分别建立sah体内及体外模型,运用western-blotting、real-timepcr等技术观察sah后组织、原代神经元及培养基中prx2在各个时程表达水平的变化,以验证sah后神经元中prx2表达水平是否上升及被释放至胞外。然后,运用western-blotting、elisa及细胞活性检测(cck8)等技术对胞外prx2对小胶质细胞(bv2)的激活(tlr4、myd88及nf-κb表达上调)、炎性因子白介素6(interleukin-6,il-6)、肿瘤坏死因子α(tumornecrosisfactor-α,tnf-α)的释放及对原代神经元的损伤等作用进一步研究,阐述胞外prx2在实验性sah后早期脑损伤的作用机制。最后,回归临床,收集行髋关节置换术及sah患者的csf,将csf浓缩50倍后用于western-blotting观察prx2的表达,然后分析csf中prx2水平与hunt-hess分级的关系。结果:体内及体外实验结果均表明:prx2在脑组织中仅神经元广泛表达,并在红细胞中含量丰富。sah后蛛网膜下腔中的红细胞裂解、其胞内prx2将进入csf中,而神经元中Prx2的m RNA水平早期迅速上调、蛋白表达随后也明显上升,随着神经元损伤、死亡、其胞内Prx2也被释放进入培养基中,这两者为胞外Prx2的来源。兔枕大池注射红细胞裂解液实验显示胞外Prx2表达与炎性因子含量正相关,并可能参与早期脑损伤作用中。随后进一步研究发现,胞外Prx2既可以直接促进神经元死亡、凋亡外,也可以通过激活TLR4/NF-κB通路、促进炎性因子释放,明显加剧神经元损伤,后者作用更为明显。最后,临床实验表明SAH后第3天患者CSF中Prx2含量升高,并与Hunt-Hess分级呈正相关。结论:本课题通过体内、体外实验再次证实:Prx2在脑组织中仅神经元广泛表达,并在红细胞中含量丰富。SAH后胞外Prx2含量上升,除直接促进神经元死亡外,更为重要的是通过激活TLR4/NF-κB通路、促进炎性因子释放,进一步加剧神经元损伤。在临床上,CSF中Prx2含量也可作为SAH患者病情判断及预后分析的可靠标志物。本课题的发现对SAH后早期脑损伤的复杂机制做了进一步地补充、完善,同样也提示通过干预蛛网膜下腔中红细胞的裂解及胞外Prx2信号通路可能会提高SAH患者的治疗效果。
[Abstract]:Background: subarachnoid hemorrhage (subarachnoid hemorrhage, SAH) is one of the three cerebrovascular diseases, with high mortality and high disability rate. A large number of red cells and their cracking products are often left in the subarachnoid space after severe.SAH. These substances can trigger a series of complicated pathophysiological changes, including brain edema, oxidative stress, inflammatory reaction, and fine effects. Cytotoxicity, etc., and these changes interlace, interact together, and eventually lead to nerve cell damage. The.Peroxiredoxin protein family (peroxiredoxins, Prxs) is a common type of eukaryotic cells, which act as antioxidants to remove the action of intracellular peroxides and mediate signals that regulate the level of hydrogen peroxide. The pathway, and participation in the development of many diseases. Further studies have found that Prxs exocytosis loses its original cell protection function and plays a role change. For example, when cerebral ischemic stroke occurs, Prxs releases to the extracellular and starts a serious inflammatory response. The mechanism can be achieved through Toll like receptor 2 (Toll-like receptor 2, TLR2) and Toll Like receptor 4 (Toll-like receptor 4, TLR4), the activation of nuclear factor kappa B (nuclear factor- kappa B, NF- kappa B) pathway activates the damage related molecular model (damage-associated molecular pattern,) type inflammatory response, which leads to the aggravation of nerve cell damage, accompanied by the progressive release, the enlargement of the proinflammatory cascade, and the eventual severe damage junction. Peroxiredoxin 2 (Prx2), one of the six members of the Prxs family, is third (second only to hemoglobin) in red blood cells, and is also widely expressed in neurons. Because of Prx2, a unique cell distribution point, it may play an extremely important role in EBI after SAH. The reason is that red blood cells and their cracking products are important injury factors of EBI after SAH, activating many damage pathways, and neurons are the final target cells of these damage pathways, which are interlaced and superimposed. Therefore, we hypothesized that when SAH occurs, the red cells in the subarachnoid cavity are gradually broken down due to aging or swelling and other factors. Prx2; neurons in brain tissue are damaged by ischemia, inflammatory reaction and other factors, and necrosis will release Prx2, causing cerebrospinal fluid, Prx2 (i.e., extracellular Prx2) expression abnormal increase of.-2- these extracellular Prx2 may also initiate, but may also initiate the damp type inflammatory reaction and aggravate the nerve. Further damage, accompanied by further release of Prx2, the role of cascade amplification, and eventually play an important role in the early brain damage after SAH. Methods: this subject first used Western-blotting technology to observe the expression of Prx2 in red blood cells, and then using immunofluorescence technique to observe the contents of Prx2 in various brain cells from the body and in vitro. Prx2 was used to verify whether the brain tissue was only expressed on the neurons. Then, the experimental subarachnoid hemorrhage model of large animals was established by the single injection of blood in the big pool of the rabbit pillow. Western-blotting, enzyme-linkedimmunosorbentassay (ELISA) technique was used to detect the erythrocyte lysate group and the red cell suspension group, and the control group was used as the control group. The content of Prx2 and inflammatory factors (IL-6, tnf- alpha) in Prx2 and cerebrospinalfluid (CSF) in the middle brain tissue. The early brain injury and basilar artery spasm were observed by immunohistochemistry, Nissl staining, he staining and other techniques. It was preliminarily concluded that extracellular Prx2 might be involved in early brain injury. Secondly, the internal and external models of SAH were established respectively. Western-blotting, real-timepcr and other techniques were used to observe the changes in the expression level of Prx2 in the tissues, the primary neurons and the culture medium in each time history, in order to verify whether the Prx2 expression level in the SAH neurons increased and was released to the extracellular. Then, the Western-blotting, ELISA and cell activity detection (CCK8) and other techniques were applied to the extracellular Prx2. The activation of microglia (BV2) (TLR4, MyD88 and nf- kappa B expression), the release of inflammatory factor interleukins 6 (interleukin-6, IL-6), tumor necrosis factor alpha (tumornecrosisfactor- a, tnf- alpha) and the damage to primary neurons, the mechanism of the action of extracellular Prx2 in the early stage of brain injury after experimental SAH was explained. Return to clinical, collect the hip arthroplasty and the CSF of SAH patients, concentrate 50 times the concentration of CSF in Western-blotting to observe the expression of Prx2, and then analyze the relationship between Prx2 level and Hunt-Hess grading in CSF. Results: in vivo and in vitro experimental results all show that Prx2 is widely expressed in the brain tissue, and the content of.Sah is rich in red cells after.Sah. The erythrocyte lysis in the subarachnoid space, its intracellular Prx2 will enter into CSF, and the m RNA level of Prx2 in the neuron is rapidly up-regulated at the early stage, and the protein expression then increases obviously. With the neuron damage, the intracellular Prx2 is released into the culture medium, both of which are the source of the extracellular Prx2. The rabbit pillow big pool is injected with the erythrocyte lysate experiment. The expression of extracellular Prx2 is positively related to the content of inflammatory factors and may be involved in early brain damage. Further studies have found that extracellular Prx2 can directly promote neuronal death and apoptosis, and can also stimulate the release of inflammatory factors by activating the TLR4/NF- kappa B pathway, which obviously aggravates the neuronal damage. The latter is more obvious. After the clinical experiment, the clinical experiment showed that the content of Prx2 in CSF increased in third days after SAH and was positively correlated with the Hunt-Hess classification. Conclusion: the subject through in vivo, in vitro experiments again confirmed that Prx2 was only widely expressed in the brain tissue, and the content of Prx2 increased after the rich.SAH in the red cells, which was more serious in addition to the direct promotion of neuronal death. It is necessary to activate the TLR4/NF- kappa B pathway to promote the release of inflammatory factors and further aggravate the neuron damage. In clinical, the Prx2 content in CSF can also be used as a reliable marker for the diagnosis and prognosis of SAH patients. The findings of this study further complement and improve the complex mechanism of early brain injury after SAH. Interfering with the lysis of erythrocytes and the extracellular Prx2 signaling pathway in the subarachnoid space may improve the therapeutic effect of SAH patients.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.35

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