内皮细胞来源外泌体对缺血再灌注损伤后神经保护作用的研究

发布时间：2018-03-28 02:31

本文选题：外泌体　切入点：缺血再灌注损伤　出处：《南昌大学》2017年硕士论文

【摘要】：目的在脑缺血的治疗中,远程缺血后处理(Remote ischemia postconditioning,RIP)能减少缺血再灌注(ischemia-reperfusion,I/R)损伤,然而,其机制未明。在本研究中,我们推测RIP对脑I/R损伤的神经保护作用是由内皮细胞释放的外泌体所介导的,探讨外泌体对人SH-SY5Y神经细胞I/R损伤后的影响。方法1.第一步建立大鼠右侧大脑中动脉缺血再灌注模型、远程缺血后处理模型,采用TTC染色、HE染色、免疫组织化学染色检测大鼠大脑组织,评估损伤程度。2.第二步建立人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)缺氧再灌注损伤模型(HUVEC I/R)及人SH-SY5Y神经细胞细胞缺氧再灌注模型(SH-SY5Y I/R),流式细胞术检测细胞凋亡情况,确定缺氧再灌注(I/R)时间;分别提取动物血清及HUVECs培养上清液中的外泌体(Exosome),电镜鉴定外泌体形态,粒径分析外泌体数量;采用免疫组化染色检测各组大鼠脑海马组织中外泌体标记蛋白CD63,HSP70,TSG101的表达量变化;Western blot与流式细胞仪检测血清中Exosome特异性蛋白CD63、HSP70和TSG101。3.第三步研究HUVEC来源外泌体对I/R损伤后的SH-SY5Y细胞增殖、凋亡、细胞周期、迁移及侵袭能力的影响,同时,检测凋亡相关分子Caspase-3、Bax和Bcl-2的表达。结果1、成功建立大鼠右侧大脑中动脉缺血再灌注模型、远程缺血后处理模型;并检测再灌注24h后大鼠大脑梗死程度:a.TTC染色结果提示缺血后处理(RIP)组大鼠大脑缺血梗死体积明显较手术(Model)组减少,假手术组(Sham)无梗死;b.HE染色结果显示假手术组大脑海马解剖结构完整,饱满,无明显凋亡;RIP组细胞萎缩,出现凋亡;Model组细胞萎缩及凋亡程度均较RIP组组明显加重;c.免疫组化染色结果提示RIP组和Model组大鼠大脑海马组织凋亡率较Sham组显著增高,且RIP组凋亡率低于Model组。2、成功建立HUVEC I/R损伤模型及SH-SY5Y I/R损伤模型,确定I/R时间为6h/24h;采用超速离心法成功提取血清及HUVECs培养上清液Exosome:a.免疫组化染色结果表明,Sham,Model,RIP各组大鼠脑海马组织中外泌体标记蛋白CD63,HSP70,TSG101的表达量无明显变化;b.透射电镜及粒径分析结果提示,外泌体形态呈圆形或椭圆形,直径在40-100nm之间,RIP组外泌体数量较Model组显著增高;c.Western Blot结果显示,各组血清中CD63,HSP70,TSG101蛋白均有表达,但RIP组较Sham,Model组表达量显著上升;流式细胞术结果表明,各组血清外泌体均表达CD63,HSP70,TSG101蛋白,且RIP组表达量较其余两组显著增高。3、CCK8实验及Edu实验结果提示HUVECs来源外泌体能有效减轻I/R损伤对SH-SY5Y细胞活力和增殖能力的影响。4、HUVECs来源外泌体对I/R损伤后SH-SY5Y细胞凋亡和细胞周期的影响:a.免疫荧光、流式细胞术、Hochest 33258实验结果表明I/R损伤后细胞凋亡显著增加,加入Exosome后能够有效减轻细胞凋亡;b.流式细胞术检测细胞周期结果提示:I/R损伤后周期阻滞在G0/G1期的SH-SY5Y细胞显著增多,S期细胞比例显著降低;与SH-SY5Y I/R组比较.SH-SY5Y I/R+Exo组在G0/G1期的细胞比例降低,S期细胞比例增高。5、HUVECs来源外泌体能有效恢复I/R损伤后SH-SY5Y细胞侵袭和迁移能力。6、qPCR及Western blot结果提示I/R损伤后细胞内Bax、Caspase-3的表达水平显著增高,Bcl-2表达水平显著降低;在Exosome处理后,I/R损伤后细胞内Bax、Caspase-3的表达水平被下调,Bcl-2表达水平被上调。结论1、RIP显著减少大鼠大脑缺血海马组织凋亡改变及缺血梗死体积。2、RIP增加大鼠血清外泌体数量及CD63,HSP70和TSG101的表达量。3、HUVECs来源外泌体能有效减轻I/R损伤对SH-SY5Y细胞活力和增殖能力的影响;HUVECs来源外泌体能有效恢复I/R损伤后SH-SY5Y细胞侵袭和迁移能力;并能够促进SH-SY5Y内相关抗凋亡基因的表达,提示外泌体有望成为治疗脑卒中的有效治疗手段之一。
[Abstract]:In the treatment of cerebral ischemia in remote ischemic postprocessing (Remote ischemia postconditioning, RIP) can reduce ischemia reperfusion injury (ischemia-reperfusion, I/R), however, its mechanism is not clear. In this study, we hypothesized that neuroprotective RIP on cerebral I/R injury with exosomes released by endothelial cells. Mediated, to explore the influence of exosomes on SH-SY5Y cells induced by I/R. Methods 1. first step to establish the rat right middle cerebral artery ischemia reperfusion model, processing model of remote ischemia by TTC staining, HE staining, staining of brain tissue of rats was detected by immunohistochemistry, evaluate the damage degree of.2. second the establishment of human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVECs) anoxia reperfusion injury model (HUVEC I/R) and SH-SY5Y cell hypoxia reperfusion model (SH-SY5Y I/R), flow cytometry Was used to detect cell apoptosis, hypoxia reperfusion time (I/R); were extracted from the serum and HUVECs culture supernatant of animal exosomes in electron microscopy (Exosome), identification of exosome exosome quantity morphology, particle size analysis; detected by immunohistochemical staining in hippocampus of rats brain and urinary marker CD63 protein, HSP70, expression of TSG101; Western and blot flow cytometry Exosome specific protein in the serum CD63, HSP70 and TSG101.3. third step HUVEC source of exosomes on SH-SY5Y cell proliferation, I/R injury and apoptosis, cell cycle, migration and invasion ability at the same time, the detection of apoptosis related molecular Caspase-3, the expression of Bax and Bcl-2. The results of the 1, successfully established the rat right middle cerebral artery ischemia reperfusion model, processing model and remote ischemia; detection of 24h after reperfusion in rats cerebral infarction: a.TTC staining Prompt ischemic postprocessing (RIP) rats cerebral ischemia infarct volume compared with surgery (Model) group, sham operation group (Sham) without infarction; b.HE staining results showed that the sham operation group of hippocampal anatomy complete, full, no obvious apoptosis; RIP cells atrophy, a group of cell atrophy and apoptosis; the degree of apoptosis was significantly higher than that of Model group RIP group increased; C. immunohistochemical staining showed that RIP group and Model group rat hippocampus apoptosis rate was significantly higher than that in Sham group, and the apoptosis rate of RIP group was lower than that of Model group.2, successfully established HUVEC I/R damage model and SH-SY5Y I/R damage model, determine the I/R time is 6h/24h; by ultracentrifugation successfully extracted the serum and HUVECs culture supernatant of Exosome:a. immunohistochemical staining showed that Sham, Model, HSP70 and RIP groups in hippocampus were foreign body urinary marker protein CD63, and the expression of TSG101 had no obvious change; B. transmission Electron microscopy and particle size analysis results suggest that exosome shapes were round or oval, with a diameter between 40-100nm, RIP group of exosome number was significantly higher than that in Model group; c.Western Blot showed that HSP70 CD63 in serum, the expression of TSG101 protein was RIP, but compared with group Sham, expression of Model group increased significantly; flow cytometry results showed that CD63 was expressed in the serum exosomes HSP70, TSG101 protein, and the expression of RIP group was significantly higher than that of other two groups.3, CCK8 and Edu experimental results indicate that HUVECs can effectively reduce the source of urinary I/R damage effects on SH-SY5Y cell viability and proliferation of.4, HUVECs source exosomes on SH-SY5Y cell apoptosis after I/R damage and cell cycle: A. immunofluorescence, flow cytometry, Hochest 33258 experimental results show that the apoptosis of I/R after injury significantly increased after adding Exosome can effectively reduce cell apoptosis; The B. flow cytometry showed that I/R after injury in G0/G1 phase arrest of SH-SY5Y cells increased significantly, the proportion of cells in S phase decreased significantly; and the SH-SY5Y I/R group.SH-SY5Y I/R+Exo group decreased the proportion of cells in G0/G1 phase, the cell ratio of S increased.5, HUVECs source can effectively restore I/R SH-SY5Y secretion after the injury of cell invasion and migration ability of.6, qPCR and Western blot showed that I/R damage Bax in cells, the expression level of Caspase-3 was significantly increased, the expression level of Bcl-2 was significantly decreased; after Exosome treatment, Bax cells within I/R after injury, the expression level of Caspase-3 was decreased, the expression level of Bcl-2 was increased. Conclusion 1. RIP was significantly reduced in rat cerebral ischemia hippocampus apoptosis and infarct size of.2, RIP increased serum exosome quantity and CD63 expression of.3 HSP70 and TSG101 HUVECs, source of excretion can effectively Reduce the I/R damage effects on SH-SY5Y cell growth and proliferation; HUVECs can effectively restore the I/R source of urinary SH-SY5Y cell invasion and migration ability after injury; and can promote the expression of SH-SY5Y in anti apoptosis gene, suggesting that exosomes in the treatment of stroke is expected to become the effective means of treatment.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743

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