星形胶质细胞Kir6.1敲除通过增强内质网应激加重小鼠脑缺血再灌注损伤

发布时间：2018-06-10 09:19

本文选题：Kir6.1/K-ATP + 星形胶质细胞　；参考：《南京医科大学》2017年硕士论文

【摘要】：脑卒中是一种发病迅速的脑血液循环障碍性疾病,致残率和致死率高,严重威胁人类健康。脑卒中主要包括缺血性脑卒中和出血性脑卒中,其中缺血性脑卒中即脑缺血,是以脑循环的血流量降低为特征的疾病,临床约80%的脑卒中患者为缺血性脑卒中。目前临床仍以重组组织型纤溶酶原激活剂治疗为主,但由于疗时间窗短和出血的危险限制了其应用。近年来旨在寻找神经保护药物治疗脑卒中的临床试验几乎均以失败告终。究其原因,现有研究往往局限于单一神经元的保护,而忽视了由神经元、星形胶质细胞、内皮细胞共同组成的神经血管单元的研究。因此,发展新的脑卒中治疗策略对于脑卒中患者尤为迫切。星形胶质细胞是中枢神经系统数量最多的细胞,是神经血管单元的重要组分之一,为神经元正常发挥功能提供保障,具有多种重要的生物学功能,包括支持、隔离、参与突触形成、转运神经递质、调节神经元物质代谢、维持离子稳态、介导信号转导等。近年来星形胶质细胞在脑缺血中的重要性逐渐被认识。星形胶质细胞在脑缺血中发生增殖、活化,特异性标记物胶原纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)表达上调。星形胶质细胞在缺血状态下耐受力较强,在脑缺血中具有双重作用:既可以产生释放谷胱甘肽和神经营养因子、生成促红细胞生成素等保护神经元,也会以释放炎症因子、减少缝隙连接和兴奋性氨基酸的再摄取等方式加重神经元损伤。因此,明确星形胶质细胞在脑缺血中的作用及其机制,有效利用星形胶质细胞对神经元的保护作用,降低损害效应,将成为脑缺血治疗的新思路,对于脑缺血的治疗具有重要的科学意义。ATP 敏感性钾通道(ATP-sensitive potassium channel,K-ATP)是一类特殊非电压依赖性的钾离子通道,耦联细胞代谢和电活动,通道的开放或关闭主要受胞内ATP/ADP比值的影响。1K-ATP通道是由内向整流钾通道(inwardly rectifying potassium channel,Kir)和磺酰脲受体(sulphonylurea receptor,SUR)亚单位以4:4组合形成的异构八聚体。Kir亚基主要包括Kir6.1和Kir6.2,其中Kir6.1亚基在脑内主要表达于星形胶质细胞、小胶质细胞和神经干细胞。本实验室前期研究发现Kir6.1/K-ATP通道与缺血性脑卒中发生发展相关,Kir6.1基因敲减加重小鼠脑缺血再灌注损伤,而且K-ATP通道开放剂减轻缺糖缺氧/复糖复氧(Oxygen-glucose deprivation/reperfusion,OGD/R)引起的星形胶质细胞损伤,提示星形胶质细胞Kir6.1/K-ATP通道可能参与了缺血性脑卒中的发生发展。本文应用星形胶质细胞Kir6.1条件敲除小鼠制备tMCAO(transient middle cerebral artery occlusion,tMCAO)模型,研究星形胶质细胞Kir6.1敲除对小鼠脑缺血再灌注急性期损伤的影响,并培养原代星形胶质细胞,观察Kir6.1表达下调对OGD/R诱导的星形胶质细胞损伤和凋亡的影响,进一步阐明星形胶质细胞Kir6.1/K-ATP通道对脑缺血再灌注损伤后内质网应激、自噬和凋亡的调节及机制。研究结果表明,星形胶质细胞Kir6.1敲除加重tMCAO引起的小鼠局灶性脑缺血再灌注损伤,增强缺血半影区内质网应激,上调自噬和凋亡水平;Kir6.1表达下调加重OGD/R引起的原代星形胶质细胞损伤,增强细胞的内质网应激,增加自噬和细胞凋亡;内质网应激阻断剂4-苯基丁酸(4-Phenylbutyric acid,4-PBA)可以逆转Kir6.1表达下调引起的星形胶质细胞损伤加重。上述结果表明星形胶质细胞Kir6.1/K-ATP通道通过调节内质网应激介导的细胞自噬和凋亡,在缺血性脑损伤中发挥重要的作用。目的:研究星形胶质细胞Kir6.1/K-ATP通道在小鼠脑缺血再灌注损伤中的作用及机制方法:1)应用Cre-Loxp系统构建星形胶质细胞Kir6.1特异性敲除小鼠。采用 2 月龄对照型(Control,CTL,KCNJ8f/f)及敲除型(Conditonalknockout,CKO,KCNJ8f/fGFAPCre)雄性小鼠,使用线栓法制备小鼠tMCAO模型,缺血1 h后再灌,于再灌后24 h对小鼠进行神经功能评分,处死小鼠并对脑梗死体积、脑含水量进行测量,探讨星形胶质细胞Kir6.1对小鼠大脑局灶短暂性缺血再灌注(tMCAO)损伤的影响;2)制备小鼠tMCAO模型,于再灌注24 h和72 h后将小鼠灌注后脑组织取材,进行冰冻组织切片,应用免疫组化技术研究星形胶质细胞Kir6.1敲除对脑缺血后神经血管单元各组分(神经元、星形胶质细胞、小胶质细胞、血管基底膜等)的影响;3)制备小鼠tMCAO模型,于再灌注24 h后免疫印迹(Western Blot,WB)法检测缺血半影区内质网应激相关蛋白、自噬相关蛋白和凋亡相关蛋白的表达;4)培养原代星形胶质细胞,转染Kir6.1 siRNA,OGD5h,复糖复氧24h,采用WB的方法检测内质网应激、凋亡相关蛋白表达。5)给予内质网应激抑制剂4-PBA,OGD5h,复糖复氧24h,应用MTT法检测细胞活力;采用Annexin-V和PI双染流式细胞仪检测凋亡率;结果:1)星形胶质细胞Kir6.1敲除加重tMCAO模型小鼠神经运动功能障碍,增大梗死体积,加重脑水肿;2)星形胶质细胞Kir6.1敲除加重缺血皮层半影区神经元的丢失,增强星形胶质细胞和小胶质细胞的增殖活化,破坏基底膜完整性;3)星形胶质细胞Kir6.1敲除加剧缺血半影区内质网应激,增加自噬和凋亡相关蛋白的表达;4)转染Kir6.1 siRNA加剧OGD/R引起的星形胶质细胞内质网应激和凋亡;5)Kir6.1表达下调加剧OGD/R引起的星形胶质细胞的损伤,增加细胞凋亡,4-PBA逆转星形胶质细胞Kir6.1表达下调引起的细胞损伤和凋亡。结论:星形胶质细胞Kir6.1敲除促进内质网应激介导的凋亡加重脑缺血再灌注损伤。综上所述,本文工作的创新之处在于:揭示星形胶质细胞Kir6.1/K-ATP通道通过调节内质网应激介导的凋亡发挥脑缺血损伤保护作用。
[Abstract]:Cerebral apoplexy is a rapid onset of cerebral blood circulation disorder with high morbidity and mortality, which seriously threatens human health. Cerebral apoplexy mainly includes ischemic stroke and hemorrhagic stroke, among which ischemic stroke is cerebral ischemia, which is characterized by the decrease of blood flow in the brain circulation, and about 80% of patients in the clinic are clinically Ischemic stroke. Currently, recombinant tissue type plasminogen activator is still the main treatment, but its application is limited due to the short time window and the risk of bleeding. In recent years, the clinical trials aimed at finding neuroprotective drugs for stroke have almost failed. Therefore, the development of a new stroke treatment strategy is particularly urgent for stroke patients. Astrocytes are the most important cells in the central nervous system, one of the important components of the neurovascular unit, and the nerve yuan Zheng Zheng. It has many important biological functions, including supporting, isolating, participating in synapse formation, transporting neurotransmitters, regulating the metabolism of neurons, maintaining ionic homeostasis, and mediating signal transduction. In recent years, the importance of astrocytes in cerebral ischemia is gradually recognized. Astrocytes are in the middle of cerebral ischemia. The expression of Glial fibrillary acidic protein (GFAP) is up regulated by the proliferation, activation and specific markers of collagen fibrillary acidic protein (GFAP). Astrocytes have a strong tolerance in ischemic state, and have double effects in cerebral ischemia: it can produce both glutathione and God management factor, and produce erythropoietin and other protective neurons. It will also aggravate neuronal damage by releasing inflammatory factors, reducing gap junctions and reuptake of excitatory amino acids. Therefore, it is a new idea for the treatment of cerebral ischemia to clarify the role and mechanism of astrocytes in cerebral ischemia and to effectively utilize the protective effects of astrocytes on neurons and reduce damage effects. The treatment of cerebral ischemia has important scientific significance, the.ATP sensitive potassium channel (ATP-sensitive potassium channel, K-ATP) is a special type of non voltage dependent potassium channel, coupled with cell metabolism and electrical activity, the opening or closing of the channel is mainly influenced by the intracellular ATP/ADP ratio, and the.1K-ATP channel is an endogenous rectifier potassium channel (inwardl). The Y rectifying potassium channel, Kir) and the sulfonylurea receptor (sulphonylurea receptor, SUR) subunits formed by the 4:4 combination of the isomeric eight polymer.Kir subunits mainly include Kir6.1 and Kir6.2, which are mainly expressed in astrocytes, microglia and neural stem cells in the brain. The TP channel is associated with the development of ischemic stroke, and Kir6.1 gene knockout aggravates the cerebral ischemia reperfusion injury in mice, and the K-ATP channel opener reduces the astrocyte damage caused by the lack of glucose hypoxia / reoxygenation (Oxygen-glucose deprivation/reperfusion, OGD/R), and the Kir6.1/K-ATP channel of astrocytes may be involved. The development of ischemic stroke. In this paper, tMCAO (transient middle cerebral artery occlusion, tMCAO) model of astrocyte Kir6.1 knockout mice was used to study the effect of Kir6.1 knockout on the acute stage of cerebral ischemia reperfusion injury in mice, and the primary astrocytes were cultured, and the expression of Kir6.1 was observed under Kir6.1. The effect of OGD/R induced astrocyte damage and apoptosis, and further elucidate the regulation and mechanism of endoplasmic reticulum stress, autophagy and apoptosis after cerebral ischemia reperfusion injury by Kir6.1/K-ATP channel in astrocytes. The results show that Kir6.1 knockout in astrocytes aggravates the focal cerebral ischemia reperfusion injury in mice caused by tMCAO Injury, enhanced endoplasmic reticulum stress in the ischemic penumbra, up-regulated the level of autophagy and apoptosis; Kir6.1 expression down-regulation aggravated OGD/R induced astrocyte damage, enhanced endoplasmic reticulum stress, increased autophagy and apoptosis; endoplasmic reticulum stress blocker 4- phenyl butyric acid (4-Phenylbutyric acid, 4-PBA) could reverse the downregulation of Kir6.1 expression The results show that the Kir6.1/K-ATP channel of star glial cells can play an important role in ischemic brain injury by regulating endoplasmic reticulum stress mediated autophagy and apoptosis. Objective: To study the role and mechanism of astrocyte Kir6.1/K-ATP channel in cerebral ischemia reperfusion injury in mice. Method: 1) the Cre-Loxp system was used to construct Kir6.1 specific knockout mice of astrocytes. 2 month old control (Control, CTL, KCNJ8f/f) and knockout (Conditonalknockout, CKO, KCNJ8f/fGFAPCre) male mice were used to prepare the mice tMCAO model using the thread thrombus method. The blood deficiency was 1 h after reperfusion. The neurological function score of the mice was scored in 24 h after reperfusion. The mice were killed and the volume of cerebral infarction and the water content of brain were measured. The effects of astrocyte Kir6.1 on cerebral focal transient ischemia and reperfusion (tMCAO) injury in mice were investigated. 2) the mouse tMCAO model was prepared. After reperfusion of 24 h and 72 h, the brain tissue of the mice was taken from the cerebral tissue, and the frozen tissue sections were sectioning. The effect of Kir6.1 knockout on astrocytes (astrocytes, astrocytes, microglia, basilar membrane and so on) after cerebral ischemia; 3) to prepare tMCAO model in mice, and to detect the endoplasmic reticulum stress related protein, autophagy related protein and withering in the penumbra of the penumbra after 24 h reperfusion. Expression of apoptosis related protein; 4) cultured primary astrocytes, transfection Kir6.1 siRNA, OGD5h, reoxygenation 24h, WB method to detect endoplasmic reticulum stress, apoptosis related protein expression.5), give endoplasmic reticulum stress inhibitor 4-PBA, OGD5h, reoxygenation 24h, use MTT method to detect cell viability; Annexin-V and PI double dye flow cytometry was used. The results were as follows: 1) 1) astrocyte knockout aggravated the neuromotor dysfunction in tMCAO model mice, increased infarct volume and aggravated brain edema; 2) Kir6.1 knockout in astrocytes aggravated the loss of neurons in the shadow area of the ischemic cortex, enhanced the proliferation and activation of astrocytes and microglia, and destroyed the integrity of the basilar membrane; 3) Kir6.1 knockout in astrocytes aggravated the endoplasmic reticulum stress in the ischemic penumbra and increased the expression of autophagy and apoptosis related proteins; 4) transfection of Kir6.1 siRNA aggravated astrocyte endoplasmic reticulum stress and apoptosis induced by OGD/R; 5) down regulation of Kir6.1 increased the damage of astrocytes caused by OGD/R, increased apoptosis, and reversed the astrocyte of 4-PBA. Conclusion: Kir6.1 knockout in astrocytes promotes endoplasmic reticulum stress mediated apoptosis and aggravates cerebral ischemia reperfusion injury. To sum up, the innovation of this work is to reveal the apoptosis mediated by the regulation of endoplasmic reticulum stress in astrocytes Kir6.1/K-ATP channels. The protective effect of cerebral ischemia injury.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3

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