脂肪间充质干细胞通过调节未折叠蛋白反应、减轻炎症反应改善脑梗死大鼠的神经功能

发布时间：2018-04-16 04:12

  本文选题：脑梗死 + 脂肪间充质干细胞　； 参考：《吉林大学》2017年博士论文

【摘要】：研究背景:脑梗死是神经内科的常见病,多发病。在世界范围内,它是导致死亡和致残的第二位原因。而在中国,它是导致死亡和成人致残的第一位原因。每年,至少有150万新发的脑梗死患者,他们中将近30%遗留有持久的严重的残疾。脑梗死已经是社会和家庭的主要负担。静脉溶栓和机械取栓是目前仅有的被FDA批准的急性脑梗死的治疗方法。但是,由于时间窗较短和有出血性转化等风险,仅有少数的患者能从中受益。因此,迫切需要一个新的疗法来治疗脑梗死。干细胞是近几年临床研究的热点。尤其是间充质干细胞,大量基础实验均证实了间充质干细胞的安全性和有效性,因此现已被批准用于临床研究。间充质干细胞科来源于骨髓、脂肪、脐带血等。虽然骨髓间充质干细胞是目前研究和应用最多的细胞,但是,实验表明,脂肪间充质干细胞和骨髓间充质干细胞具有相同的生物学特性,而且,脂肪间充质干细胞比骨髓间充质干细胞具有更多的优势,它具有来源丰富,取材简单,易于培养等特点。在缺血性脑卒中治疗中,具有调节炎症、血管再生及抗凋亡等作用,能够明显的改善血脑屏障的通透性,减少梗死体积,改善神经功能缺损症状。因此,脂肪间充质干细胞移植治疗有望成为临床上治疗急性脑梗死的最为理想的细胞。越来越多的证据表明,脂肪间充质干细胞能够改善缺血再灌注损伤大鼠的神经功能,但是,其作用机制一直比较复杂。了解其作用机制有助于基础向临床的转化。目前的研究表明,脂肪间充质干细胞在脑梗死的治疗中具有抗炎、抗凋亡、促进血管再生、神经再生等作用。尤其是急性期,抗炎抗凋亡的作用尤为显著。大量研究炎症与缺血再灌注损伤后的血脑屏障的破坏是密不可分的。未折叠蛋白反应(UPR)可以促进脑梗死后的细胞凋亡。因此,本实验选用ADMSCs用于治疗急性脑梗死大鼠,并通过对炎症介质、凋亡反应、血脑屏障透通性、UPR调节的观察来探讨ADMSCs可能的作用机制。目的:(1)观察ADMSCs的表面标志物及其向脂肪细胞和成骨细胞的定向分化能力;(2)观察ADMSCs治疗后血脑屏障的变化;(3)观察ADMCs对缺血再灌注大鼠脑梗死体积的影响及其对神经功能缺损症状的影响;(4)观察ADMSCs对脑梗死后小胶质细胞增生和炎症介质释放的影响;(5)观察ADMSCs对凋亡及UPR调节的影响。根据这些结果进一步证实ADMSCs对急性脑梗死治疗的有效性及其作用机制。实验方法:(1)选取250g-300g的SD大鼠作为实验对象,制作大脑中动脉闭塞(MCAO)-2小时缺血再灌注模型;(2)提取分离并培养脂肪间充质干细胞,选用P3代细胞作为实验用细胞,在细胞注射前30分钟,用流式细胞仪检测法检测ADMSCs的表面标志物。同时,将P3代细胞用成脂和成骨诱导液进行诱导,21天后采用油红O染色和茜素红染色的方法鉴定ADMSCs向脂肪细胞和成骨细胞的分化能力;(3)将MCAO大鼠随机分为3组,sham组,MCAO组和MCAO+ADMSCs组,在造模后0,12小时,24小时分别给予尾静脉注射等容量PBS和ADMSCs(2×106个);(4)治疗后第1天,第3天,第7天,第14天用m NSS评分法进行大鼠的行为功能测试;(5)在治疗第1天,第7天,第14天用伊文思蓝染色的方法观察大鼠血脑屏障的变化;(6)在治疗第14天处死大鼠,通过免疫荧光染色观察大鼠血脑屏障紧密连接蛋白ZO-1和Claudin-5的表达,同时观察小胶质细胞增生OX-42的表达;(7)在治疗14天,用PT-PCR技术检测炎症介质TNF-α,IL-1β,IL-6的表达;(8)通过TUNEL染色的方法观察梗死区神经细胞的凋亡;(9)通过westernblot的方法观察ADMSCs对UPR调节信号通路的GRP78,PERK,p-PERK,e IF2α,p-e IF2α,ATF4,CHOP,Bax,Bcl-2,XBP-1表达的影响。实验结果:(1)ADMSCs高度表达间充质干细胞的表面标志物CD44,CD29,CD90,而不表达造血干细胞的表面抗原CD34,CD45;(2)成脂诱导液和成骨诱导液诱导21天后,油红O染色和茜素红染色发现ADMSCs可以向成脂细胞和成骨细胞定向分化;(3)TTC染色观察到,与MCAO相比,ADMSCs治疗能明显的减少MCAO大鼠的梗死体积(P0.01);(4)m NSS评分的比较,ADMSCs组明显的降低m NSS的评分,这种改善神经功能缺损的效果从第7天开始一直持续到第14天(P0.01);(5)伊文思蓝染色显示,造模后第一天开始血脑屏障明显破坏,并随着时间的延长逐渐好转,ADMSCs治疗在第7天明显改善MCAO引起的血脑屏障的破坏;(6)PT-PCR检测发现,MCAO后梗死区的TNF-α,IL-1β,IL-6的表达明显增高(与sham组相比,P0.01),ADMSCs治疗能够明显的降低这三种炎症介质的释放(P0.01);(7)免疫荧光染色发现,MCAO组血脑屏障爱紧密连接蛋白ZO-1,Claudin-5的荧光强度明显降低(P0.01),ADMSCs治疗后能明显的增强它们荧光强度的表达(P0.01),说明ADMSCs能改善缺血再灌注损伤的血脑屏障;(8)免疫荧光染色同时发现ADMSCs治疗能减少MCAO后的OX-42的表达,两组之间具有显著差异(P0.01);(9)ADMSCs治疗能够明显的减少脑梗死及周边区域的细胞凋亡,与MCAO组具有显著差异(P0.01);(10)通过Westernblot观察UPR信号通路上的蛋白,发现ADMSCs能够明显的升高抗凋亡因子Bcl-2的表达,降低GRP78,p-PERK,p-e IF2α,ATF4,CHOP,Bax,XBP的表达,与MCAO组相比具有显著的统计学差异(P0.01)。结论:(1)ADMSCs取材简单,来源丰富,易于培养,没有免疫排斥反应,具有诱导分化的能力,是基础及临床上细胞治疗的较为理想的细胞来源。(2)多次、静脉注射ADMSCs能够明显的减少脑缺血再灌注损伤的梗死体积,改善神经功能缺损症状。(3)早期给予ADMSCs能够明显的改善缺血再灌注损伤大鼠的血脑屏障。(4)ADMSCs治疗能够减轻缺血再灌注大鼠的炎症反应,进而降低血脑屏障的通透性。(5)脑缺血再灌注损伤能够诱发内质网的UPR,促进凋亡反应。ADMSCs可能是通过调节UPR反应来降低凋亡,进而改善MCAO大鼠的神经功能症状。
[Abstract]:Background: cerebral infarction is a common disease in neurology, the disease. In the world, it is the leading cause of death and disability in second. China, it is leading cause of death and disability in adults. Every year, there are at least 150 million new cases of cerebral infarction patients, they will have a lasting legacy of nearly 30% the serious disability. Cerebral infarction is a major burden on society and family. Intravenous thrombolysis and mechanical thrombectomy is the treatment of acute cerebral infarction is currently the only approved by the FDA. However, due to a relatively short time window and the risk of hemorrhagic transformation, only a small number of patients can benefit from it. Therefore, an urgent the new therapy to treatment of cerebral infarction. Stem cells are the hotspot in recent years. Especially the clinical study of mesenchymal stem cells, a large number of basic experiments have confirmed the safety and efficacy of mesenchymal stem cells, so it has been approved by In the clinical study. Mesenchymal stem cells derived from bone marrow, umbilical cord blood, fat, etc.. Although bone marrow mesenchymal stem cells is the research and application of most cells, however, experiments show that adipose derived mesenchymal stem cells and bone marrow mesenchymal stem cells have the same biological characteristics, but also between fat mesenchymal stem cells have more advantages than the bone marrow mesenchymal stem cells, it has a rich source of material is simple, easy to culture and so on. In the treatment of ischemic stroke, with the regulation of inflammation, angiogenesis and anti apoptosis, can significantly improve the permeability of the blood-brain barrier, reduce infarct volume and improve defect the symptoms of nerve function. Therefore, adipose derived mesenchymal stem cells transplantation for the treatment of the clinical treatment of acute cerebral infarction is expected to become the most ideal cells. More and more evidence that adipose derived mesenchymal stem cells can improve ischemia reperfusion Note the damage of neurological function in rats, but its mechanism is complex. To investigate the mechanism of help to clinical translation. The present study showed that adipose derived mesenchymal stem cells in the treatment of cerebral infarction with anti-inflammatory, anti apoptosis, promoting angiogenesis, nerve regeneration and so on. Especially acute, anti-inflammatory and anti apoptosis effect is particularly significant. A lot of inflammation and ischemia reperfusion injury after the destruction of the blood-brain barrier is inseparable. The unfolded protein response (UPR) can promote cell apoptosis after cerebral infarction. Therefore, this experiment used ADMSCs for the treatment of acute cerebral infarction in rats, and the apoptosis in response to inflammation medium, blood brain barrier permeability, observe the regulation of UPR to explore the possible mechanisms of ADMSCs. Objective: (1) to observe the surface markers of ADMSCs were related to adipogenic and osteogenic differentiation ability; (2) The changes of blood brain barrier was observed after treatment with ADMSCs; (3) effect of ADMCs cerebral infarction volume in rats on ischemia reperfusion and its effect on neurological function; (4) to observe the effect of ADMSCs on proliferation of microglia and release of inflammatory mediators after cerebral infarction; (5) to observe the effect of ADMSCs on apoptosis and regulation UPR. According to these results further confirmed the effectiveness of ADMSCs for the treatment of acute cerebral infarction and its mechanism. Methods: (1) select 250g-300g SD rats as experimental object, making middle cerebral artery occlusion (MCAO) -2 hour ischemia reperfusion model; (2) adipose derived mesenchymal stem cells and culture extraction the P3 generation of cells, as in the experiment, cells were injected 30 minutes before using the surface markers of ADMSCs by flow cytometry assay. At the same time, the fat and osteogenic induction medium in induced by P3 cells, after 21 days by oil red O 鏌撹壊鍜岃寽绱犵孩鏌撹壊鐨勬柟娉曢壌瀹欰DMSCs鍚戣剛鑲�缁嗚優鍜屾垚楠ㄧ粏鑳炵殑鍒嗗寲鑳藉姡�

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