神经干细胞对缺血性卒中的保护机制研究

发布时间：2018-05-23 23:06

本文选题：缺血性卒中 + 神经干细胞　；参考：《第二军医大学》2017年博士论文

【摘要】：脑卒中主要发生在老年人群中,是老年人群致残的重要原因之一,以脑梗死为主。随着我国人口进入老龄化时代,脑梗死患病率逐年升高,严重降低了患者的生存质量和平均寿命,严重危害老年人的身体健康,给国民经济带来沉重的负担。目前临床上传统抗凝、溶栓、神经保护以及后期的康复等治疗效果有限,大部分脑卒中患者后期仍留下各种偏瘫、失语等不同程度的经功能障碍后遗症。因此,寻找新的治疗方法促进缺血性脑血管病患者神经功能的恢复已成为当今研究的重点[1],在过去的四十年里,脑卒中发病率在发达国家降低了43%,而在发展中国家却增长了100%,发生于这些国家的脑卒中死亡数约占脑卒中死亡总数的87%[2]。若没有有效干预措施,至2030年全球脑卒中死亡人数将增加至780万[3]。脑卒中患者中87%是缺血性脑梗死[4]。多种疾病均可能导致缺血性卒中,而其最重要的病因包括大动脉粥样硬化(血管),心源性脑栓塞,脑小血管病(微血管病变)。不太常见的病因是颈部动脉夹层动脉瘤、脑血管炎、凝血功能障碍性疾病、血液病等。即使在完成相关诊断检查仍有部分缺血性脑卒中患者无法明确病因。对于急性缺血性脑梗死,FDA只认证了早期溶栓作为治疗手段。但由于时间窗的限制,95%以上的患者无法得到溶栓治疗[5]。因此,探索新的、能让更多患者受益的缺血性卒中治疗策略具有极其重要的现实意义。近年来,神经干细胞(Neural stem cells,NSCs)在脑梗死治疗中的应用研究,为解决这一难题提供了新的思路和方法。国内外已有相关研究证实了NSCSs具有显著的神经保护作用[6,7]。1.在急性脑梗死的研究中,NSCs对于血管内皮细胞的保护作用尚不明确。脑微血管内皮细胞(BMECs)不仅仅是血管腔的内衬,还能分泌活性物质影响神经元的生存。我们针对NSCs能否有效修复缺血损伤的BMECs开展了进一步研究。2.我们发现NSCs可以通过直接或间接接触的方式保护缺血损伤的内皮细胞:在前期实验中,我们成功分离和培养了NSCs和BMECs;为了进一步模拟缺血损伤中内皮细胞所处的病理生理条件,我们对BMEC实施了缺糖缺氧/复糖复氧(oxygen-glucose deprivation,OGD)的类缺血再灌注损伤处理,并建立了这两种细胞的共培养模型;证实了NSCs能有效保护和修复OGD-BMECs。那么,NSCs是通过什么机制来发挥这种保护作用的呢?早期的研究认为干细胞分化和细胞融合是修复的可能机制,但是当前更多的研究对以上两种观点提出了置疑。研究发现,用NSCs处理中动脉栓塞(middle cerebral artery occlusion,MCAO)鼠模型,其功能评分得到很大改善,但GFP标记显示能够真正到达病灶分化成神经元的NSCs数量非常有限[8]。这些外源性NSCSs来源的神经元细胞无法解释小鼠功能评分的显著改善。因此,促使人们探寻NSCSs的其它可能保护机制。3.PI3K/Akt/GSK-3b/m TOR信号通路参与了代谢调节、细胞生长和存活等多种重要功能。PI3K活性形式为一种原癌基因,多种肿瘤疾病发现有PI3K的异常扩增和突变。在哺乳动物细胞中,基于结构、分布及活化机制的不同,PI3K可被分为三类,I类PI3Ks分为Ia和Ib两类[9]。IA类PI3K可被受体酪氨酸激酶激活,而Ib类PI3K则被G蛋白偶联受体激活。这些PI3K均是由调节亚基(如p85)和催化亚基(如P110)组成的异源二聚体。PI3K参与控制细胞的迁移和血管生成。PI3K产生的磷脂第二信使参与细胞内信号转导过程中的多个步骤。PI3K可磷酸化和激活Akt,将其定位在质膜上[10]。Akt是PI3K调节细胞生长和细胞迁移的主要下游靶因子。在人体内有三种亚型:Akt1、Akt2和Akt3。PI3K产生的PIP3结合Akt并导致Akt的膜聚集,并通过其PH结构域结合磷酸肌醇依赖性蛋白激酶1(PDK1),然后PDK1磷酸化Akt激酶结构域[11]。为充分激活Akt,也需PDK2磷酸化AKT羧基端调节区域(AKT1在丝氨酸473位)。一旦激活,AKT就进入细胞质和细胞核,产生一系列下游影响,如激活CREB,抑制p27,将FOXO定位于细胞质中,激活ptdins-3ps,影响P70和4EBP1的转录导致m TOR复合物的聚集和激活。m TOR调节核糖体蛋白S6激酶和真核翻译起始因子4E结合蛋白1进而引起翻译起始因子e IF4E的释放。糖原合成酶激酶-3β(GSK-3β)是一种丝氨酸/苏氨酸激酶,已有研究证明该激酶参与细胞增殖、细胞程序性死亡,胚胎发生,调节昼夜节律。GSK-3β属于保守丝氨酸/苏氨酸激酶家族,它的活性可以在Tyr残基被磷酸化后激活,而其活性会在Ser9残基部位被磷酸化后抑制,多种细胞功能的调节均有GSK-3β的参与,包括基因表达、代谢、和维持细胞骨架的完整性,GSK-3β是一种细胞生长、增殖的负性调控因子[12]。许多已知因素包括EGF,shh,IGF-1、胰岛素和Cam等均可激活PI3K/Akt通路,而PTEN,HB9等可拮抗该通路表达[7,11,13-16]。越来越多的证据显示,NSCSs与BMEC直接接触共培养对PI3K/Akt/GSK-3β/m TOR信号通路的影响可能增加了NSCs对内皮细胞功能的调节作用[9]。研究该通路在此过程中作用机制,可望为脑卒中的合理预防、逆转提供重要依据。本课题拟通过体内外的缺血缺氧模型探讨神经干细胞在卒中病变中的神经保护机制及PI3K/Akt/GSK-3β/m TOR信号通路的影响。实验方法1.原代培养神经元2.不同浓度GSK-3β抑制剂TWS119不同浓度处理细胞,MTT法检测神经元活性变化3.MAP-2荧光染色后使用Image-J软件计数神经元突触总长度变化4.Western Blot检测Mcl-1、Bcl-2、Bax凋亡通路相关蛋白表达变化5.原代培养脑微血管内皮细胞及胎鼠培养神经干细胞,将BMECs置于氧糖剥夺环境中,两种细胞使用Transwell小室进行间接共培养6.Casepase-3凋亡相关酶活性及LDH酶活性检测细胞损伤7.Western Blot及q-rt PCR检测PI3K/AKT/GSK-3信号通路表达变化8.制作大脑中动脉栓塞模型在SD大鼠中模拟缺血性脑梗,设置假手术组,NSCs治疗组,动物疾病模型组、PI3k抑制剂组进行免疫组化染色等体内实验9.行为学及TTC染色明确脑梗模型建立10.HE染色、尼氏染色观察神经干细胞移植后脑组织形态学改变11.MAP-2、GAP-43、Sinapsin-1染色观察神经元、突触重塑情况12.IBA-1、GFAP免疫组化观察小胶质细胞、星形胶质细胞激活情况反映免疫激活状态13.Beclin-1、LC-3免疫组化检测自噬活性14.检测细胞、动物模型中炎症因子表达变化15.错步实验及黏胶移除实验进行脑梗后行为学观察。结果1.MTT染色显示细胞活性在氧糖剥夺环境中显著降低,但GSK-3β抑制剂TWS119处理后神经元活性提高2.GSK-3β抑制剂TWS119增加神经元突触总长度3.GSK-3β抑制剂TWS119能增加抗凋亡蛋白Mcl-1、Bcl-2的表达,而下调促凋亡蛋白Bax4.GSK-3β抑制剂TWS119可使抗炎因子IL-10表达增加,而促炎因子IL-1β,IL-6及ICAM-1下降,炎症因子IFN-γ表达未见明显变化5.原代培养脑微血管内皮细胞及胎鼠神经干细胞使用Transwell小室进行间接共培养,其PI3K/AKT/GSK-3信号通路磷酸化水平增高,Casepase-3凋亡相关酶活性及LDH酶活性检测提示细胞损伤较单纯缺血模型细胞组减轻6.制作大脑中动脉栓塞模型在SD大鼠中模拟缺血性脑梗,设置假手术组,NSCs治疗组,动物疾病模型组进行免疫组化染色等体内实验7.大鼠行为学、TTC染色明确脑梗模型建立8.HE染色、尼氏染色观察神经干细胞注射后脑组织核固缩、核碎裂及尼氏染色缺失等病理改变有所减轻9.MAP-2、GAP-43、Sinapsin-1染色观察显示细胞存活量在NSCs治疗后明显提高10.IBA-1、GFAP免疫组化观察NSCs能降低缺血损伤引起的炎症反应细胞激活,自噬活性也降低11.NSCs治疗后,动物模型中炎症因子L-Selectin,leptin,MCP-1及TNFα表达降低,抗炎因子TIMP-1表达升高12.NSCs治疗组大鼠行为学实验中错步数及黏胶移除所需时间均有所减少13.以上变化可被PI3k抑制剂LY294002部分逆转结论抑制神经元GSK-3β对氧糖剥夺环境下的神经元产生保护作用,提高神经元活性,使神经元突触长度变长,抑制凋亡活性。机体内产生GSK-3β抑制作用的主要上游信号通路由PI3K/AKT介导,神经干细胞能激活氧糖剥夺环境下BMECs中该通路,同时通过抗凋亡、抗炎、抑制自噬等作用促进MCAO模型鼠神经功能恢复。这些作用部分被PI3K抑制剂LY294002所抵消,说明神经干细胞通过PI3K/AKT/GSK-3β信号通路参与卒中损伤的神经保护作用。
[Abstract]:Cerebral apoplexy is one of the main causes of the elderly population. It is one of the most important reasons for the disabled in the elderly. With the age of aging in China, the incidence of cerebral infarction is increasing year by year, which seriously reduces the life quality and average life span of the patients, seriously endangers the health of the elderly and brings heavy burden to the national economy. The therapeutic effect of traditional anticoagulant, thrombolytic, neuroprotective and later rehabilitation is limited, and most of the stroke patients still leave a variety of hemiplegia, aphasia and other dysfunctional sequelae. Therefore, looking for a new treatment method to promote the recovery of neural function of patients with ischemic cerebral blood tube disease has become the present research. In the past forty years, the incidence of stroke has decreased by 43% in developed countries, while in developing countries, the incidence of stroke has increased by 100% in the developing countries. The number of stroke deaths in these countries accounts for about 87%[2]. of the total number of stroke deaths in these countries without effective intervention, and the number of global stroke deaths will increase to 7 million 800 thousand [3]. stroke by 2030. 87% of the patients with ischemic cerebral infarction may cause ischemic stroke in many [4]. diseases. The most important causes include large atherosclerosis (vascular), cardiogenic cerebral embolism, and small vascular disease (microvascular disease). The most common causes are carotid artery dissecting aneurysm, cerebral vasculitis, coagulation dysfunction disease, and hematological diseases. Even in the completion of the related diagnosis, some patients with ischemic stroke are still unable to identify the cause. For acute ischemic cerebral infarction, FDA only certified early thrombolytic therapy. But because of the time window, more than 95% of the patients are unable to get thrombolytic therapy for [5].. In recent years, the application of Neural stem cells (NSCs) in the treatment of cerebral infarction has provided a new way of thinking and method to solve this problem. The relevant research at home and abroad has confirmed that NSCSs has a significant neuroprotective effect of [6,7].1. in the study of acute cerebral infarction. The protective effect of NSCs on vascular endothelial cells is not clear. Cerebral microvascular endothelial cells (BMECs) are not only the inner lining of the vascular cavity, but also secrete active substances to affect the survival of neurons. We have further studied whether NSCs can effectively repair the BMECs of ischemic damage. We found that NSCs can be directly or indirectly exposed. We successfully isolated and cultured NSCs and BMECs in the early experiments. In order to further simulate the pathophysiological conditions of endothelial cells in ischemic injury, we carried out ischemic reperfusion injury treatment for BMEC deficiency / hypoxia / reoxygenation (oxygen-glucose deprivation, OGD). A co culture model of these two cells has been established, and it has been confirmed that NSCs can effectively protect and repair OGD-BMECs.. What mechanism does NSCs use to play this protective effect? Early studies suggest that stem cell differentiation and cell fusion are possible mechanisms for repair, but more studies have questioned the above two views. It was found that the function score of the middle cerebral artery occlusion (MCAO) rat model was greatly improved with NSCs, but the GFP markers showed that the number of NSCs that could really reach the lesion differentiation into neurons was very limited and the number of the xenobiotic cells derived from exogenous NSCSs could not explain the significant improvement of the function score of the mice. Therefore, the other possible protective mechanism of NSCSs,.3.PI3K/Akt/GSK-3b/m TOR signaling pathway, is involved in metabolic regulation, cell growth and survival, such as cell growth and survival, as a proto oncogene, and a variety of tumor diseases are found to have abnormal amplification and mutation of PI3K. In mammalian cells, structure based, distribution Different from the activation mechanism, PI3K can be divided into three classes. Class I PI3Ks is divided into Ia and Ib two classes [9].IA PI3K can be activated by receptor tyrosine kinase, and Ib class PI3K is activated by G protein coupled receptors. These PI3K are heterogenous two polymers composed of regulatory subunits (such as p85) and catalytic subunits, which participate in the control of cell migration and angiogenesis. The phosphatidylcholine second messenger produced by.PI3K is involved in multiple steps in the intracellular signal transduction process.PI3K can phosphorylation and activation of Akt, which is located on the plasma membrane and [10].Akt is the main downstream target factor for PI3K regulating cell growth and cell migration. There are three subtypes in the human body: PIP3 binding Akt produced by Akt1, Akt2 and Akt3.PI3K and leading to Akt The membrane aggregates, and through its PH domain, binding the inositol phosphoric acid dependent protein kinase 1 (PDK1), and then PDK1 phosphorylated Akt kinase domain [11]. to fully activate Akt, and also the PDK2 phosphorylation of the AKT carboxyl terminal regulating region (AKT1 at the serine 473). Once activated, AKT enters the cell and nucleus, producing a series of downstream effects, such as activating CREB, Inhibition of p27, FOXO is located in the cytoplasm, activates ptdins-3ps, affects the transcription of P70 and 4EBP1, causes the aggregation of M TOR complexes and activates the.M TOR regulated ribosome S6 kinase and the eukaryotic translation initiation factor 4E binding protein 1, thus causing the release of the translation initiation factor E. The kinase, which has been proved to be involved in cell proliferation, programmed cell death, embryogenesis, and regulating the circadian rhythm of.GSK-3 beta, is a conservative serine / threonine kinase family, its activity can be activated after the phosphorylation of the Tyr residue, and its activity will be inhibited after the phosphorylation of the Ser9 residues and the regulation of a variety of cell functions. The participation of GSK-3 beta, including gene expression, metabolism, and maintenance of the integrity of cytoskeleton, GSK-3 beta is a cell growth, a negative regulator of proliferation, [12]., many known factors including EGF, Shh, IGF-1, insulin and Cam can activate PI3K/Akt pathway, and PTEN, HB9, etc. can antagonize the increasing evidence of this pathway to express [7,11,13-16].. The effect of direct contact between NSCSs and BMEC on PI3K/Akt/GSK-3 beta /m TOR signaling pathway may increase the regulatory role of NSCs on endothelial cell function and [9]. study the mechanism of this pathway in this pathway, which is expected to provide an important basis for the rational prevention and reversal of stroke. The neuroprotective mechanism of neural stem cells and the effect of PI3K/Akt/GSK-3 beta /m TOR signaling pathway in stroke disease. Experimental methods 1. primary cultured neurons 2. different concentrations of GSK-3 beta inhibitor TWS119 were treated with different concentrations of TWS119 cells, MTT assay was used to detect neuronal activity and 3.MAP-2 fluorescence staining was used to count the synapse total of neurons by Image-J software Length variation 4.Western Blot detection of Mcl-1, Bcl-2, Bax apoptosis pathway related protein expression changes 5. primary culture of brain microvascular endothelial cells and fetal rat culture neural stem cells, BMECs in oxygen deprivation environment, two cells using Transwell chamber for indirect co culture 6.Casepase-3 apoptosis related enzyme activity and LDH enzyme activity detection Cell injury 7.Western Blot and q-rt PCR detection of PI3K/AKT/GSK-3 signaling pathway expression change 8. to make cerebral artery embolism model in SD rats to simulate ischemic infarction in SD rats, set up sham operation group, NSCs treatment group, animal disease model group, PI3k inhibitor group with immunohistochemical staining in vivo and TTC staining to clear brain infarction model 10.HE staining was established. Nissl staining was used to observe the morphological changes of brain tissue after neural stem cell transplantation. 11.MAP-2, GAP-43, Sinapsin-1 staining was used to observe neurons, synaptic remodeling was observed in 12.IBA-1, GFAP immunohistochemistry was used to observe the microglia, the activation of astrocytes reflected the immune activation state 13.Beclin-1, and LC-3 immunochemistry was used to detect autophagy. Sex 14. detected cells, the expression of inflammatory factors in the animal model, the 15. error step experiment and the adhesive removal experiment to observe the post infarction behavior. Results 1.MTT staining showed that the cell activity was significantly reduced in the oxygen deprivation environment, but the activity of GSK-3 beta inhibitor TWS119 was higher than that of 2.GSK-3 beta inhibitor TWS119 to increase the neuron synapse. The total length of 3.GSK-3 beta inhibitor TWS119 could increase the expression of anti apoptotic protein Mcl-1 and Bcl-2, while the down-regulation of Bax4.GSK-3 beta inhibitor TWS119 could increase the expression of anti inflammatory factor IL-10, while the proinflammatory factor IL-1 beta, IL-6 and ICAM-1 decreased, and the expression of inflammatory factor IFN- gamma did not significantly change 5. primary cultured cerebral microvascular endothelial cells and fetal rat God. Through indirect co culture of Transwell cells through the use of stem cells, the level of phosphorylation of the PI3K/AKT/GSK-3 signaling pathway increased, the activity of Casepase-3 apoptosis related enzymes and the activity of LDH enzyme suggested that the cell damage was reduced by 6. to make the middle cerebral artery embolism model in the SD rats to simulate the ischemic cerebral infarction in the SD rats and set up the sham operation. Group, NSCs treatment group, animal disease model group with immunohistochemical staining and other in vivo experiments of 7. rats, TTC staining clear brain infarction model established 8.HE staining, Nissl staining observation of neural stem cells after injection of brain tissue nuclear pyknosis, nuclear fragmentation and Nissl staining loss and other pathological changes to reduce 9.MAP-2, GAP-43, Sinapsin-1 staining observed obvious The survival of the cells was significantly increased by 10.IBA-1 after NSCs treatment. NSCs could reduce the activation of inflammatory cells induced by ischemic injury, and the autophagy activity also reduced 11.NSCs treatment. The expression of inflammatory factors L-Selectin, leptin, MCP-1 and TNF alpha in the animal model decreased, and the expression of anti inflammatory factor TIMP-1 increased in the 12.NSCs treatment group. The time required for the number of wrong steps and the removal of sticky glue in the behavioral experiment can be reduced by more than 13., which can be reversed by the partial reversal of the PI3k inhibitor LY294002, which inhibits the protective effect of GSK-3 beta on neurons in the oxygen deprivation environment, improves the activity of neurons, increases the length of the synapse and inhibits the apoptosis activity, and produces GSK-3 in the body. The main upstream signal of beta inhibition is mediated by route PI3K/AKT. Neural stem cells can activate the pathway of BMECs in the oxygen deprivation environment, and promote the recovery of neural function in MCAO model rats by anti apoptosis, anti-inflammatory and autophagy. These effects are partly offset by PI3K inhibitor LY294002, indicating that neural stem cells pass through PI3K/AKT/G. SK-3 beta signaling pathway is involved in the neuroprotective effect of stroke injury.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.3

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