p38调控VE-Cadherin移位在热打击HUVECs高通透性中的作用

发布时间：2018-03-27 22:40

  本文选题：热打击　切入点：丝裂原活化蛋白激酶　出处：《广州中医药大学》2017年硕士论文

【摘要】：中暑(heat stroke)由于全身炎症反应(SIRS)作用,广泛存在着血管内皮(VEC)损伤,伴随血管渗漏、出血、组织水肿等病理改变,导致微循环障碍、休克与多器官功能障碍综合征(MODS)。血管通透性的改变与血管内皮的粘附连接(AJ)及血管内皮钙粘素(VE-Cadherin)的功能结构紧密相关。然而,中暑的血管通透性变化规律及其深入分子信号通路机制的研究仍有待开展。目的:1.研究热打击下人脐静脉内皮细胞(HUVECs)的通透性变化规律。2.研究p38 MAPK通路对热打击所致HUVECs通透性变化及对VE-Cadherin分布变化的影响。方法:1.热打击引起内皮细胞通透性变化及其分子机制1.1.构建体外HUVECs热打击模型,根据不同热打击温度分为对照组、39℃组、41℃组和43℃组,利用Transwell技术检测单层内皮细胞跨膜电阻值(TEER),利用荧光蛋白跨膜渗漏实验计算细胞通透系数(Pa)。TEER越低、Pa越大,细胞通透性越高;1.2.根据不同热打击时间把HUVECs分为对照组、1h组、2h组、和3h组,检测TEER,计算Pa,研究不同热打击时间对细胞通透性的影响。1.3.根据不同热打击时间把HUVECs分为对照组、0.5h组、1h组、1.5h组和2h组,利用Western Blot方法检测同一热打击温度下(43℃)HUVECs中VE-Cadherin总蛋白、膜蛋白和浆蛋白的含量与分布。2.p38 MAPK通路在热打击引起内皮细胞透通性变化中的作用2.1.构建HUVECs热打击模型,根据不同热打击时间分为对照组、0.5h组、1h组、1.5h组和2h组,Western Blot方法检测HUVECs中p38蛋白的磷酸化水平;2.2.构建HUVECs热打击模型,根据不同的处理因素分为对照组、SB组(p38抑制剂SB203580,浓度10uM)、HS组(热打击组)和SB+HS组。其中,SB组是预先在HUVECs加入SB203580并于细胞培养箱中共孵育2h,SB+HS组是在HUVECs加SB203580共孵育2h后行43℃热打击2h。检测各组TEER,计算Pa,以此表示HUVECs通透性;2.3.根据不同处理因素把HUVECs分为对照组、SB组、HS组和SB+HS组。分组处理后,通过Western blot及免疫荧光的方法检测VE-Cadherin在胞膜、胞浆中的含量及分布情况。结果:1.热打击引起内皮细胞透通性变化及其分子机制1.1.不同热打击温度下HUVECs的通透性改变:热打击下HUVECs的TEER显著降低、Pa显著增大,细胞通透性增高。随热打击温度升高,TEER逐渐降低、Pa逐渐增大;1.2.不同热打击时间下HUVECs的通透性改变:随热打击时间延长,TEER逐渐显著降低、Pa逐渐显著增大,细胞通透性增高。1.3.热打击下 HUVECs 的 VE-Cadherin 改变:1.3.1.在热打击过程中,HUVECs中VE-Cadherin蛋白总量随热打击时间延长变化无统计学意义;1.3.2.在热打过程中,HUVECs中VE-Cadherin在胞膜上含量显著减少,在胞浆中含量显著增加。2.p38 MAPK通路在热打击引起内皮细胞透通性变化中的作用2.1.早期热打击即可使HUVECs的p38磷酸化水平显著增高;2.2.p38 MAPK通路抑制剂SB203580预处理,热打击所致HUVECs的TEER降幅显著减小、Pa增幅显著减小,稳定了细胞通透性;2.3.SB203580预处理,Western Blot及免疫荧光均提示热打击引起的HUVECs中VE-Cadherin原"胞膜减少、胞浆增加"的重新分布趋势显著减轻;结论:热打击下HUVECs通透性呈温度时间依赖性增高,其VE-Cadherin随热打击发生从胞膜到胞浆的再分布。热打击早期即引起P38磷酸化,并通过p38 MAPK通路调控VE-Cadherin从胞膜到胞浆的移位,造成细胞间粘附连接的破坏,引起HUVECs通透性增高。
[Abstract]:Heatstroke (heat stroke) due to systemic inflammatory response (SIRS), there is a wide range of vascular endothelial injury (VEC), accompanied by vascular leakage, hemorrhage, edema and other pathological changes, cause microcirculation, shock and multiple organ dysfunction syndrome (MODS). The change of vascular adhesion permeability and endothelial connection (AJ) and vascular endothelial cadherin (VE-Cadherin) is closely related to the function of the structure. However, the research of vascular permeability changes and further heatstroke pathway mechanism still needs to be carried out. Objective: 1. to study the thermal impact of human umbilical vein endothelial cells (HUVECs) permeability change law of.2. research p38 MAPK pathway on HUVECs induced by heat stress permeability change and influence on VE-Cadherin distribution. Methods: 1. hot blow variation caused by the permeability of endothelial cells and the molecular mechanism of 1.1. in vitro HUVECs heat stroke model, according to the different The temperature of heat stroke divided into control group, group C 39, 41 C and 43 C group group, detected by monolayer endothelial cells Transwell transmembrane resistance (TEER), and calculate the cell permeability coefficient by fluorescent protein transmembrane leak experiment (Pa).TEER is low, Pa is higher, the higher the permeability of cells according to 1.2.; different heat attack time of the HUVECs is divided into control group, 1H group, 2h group and 3H group, detection of TEER, Pa is calculated, the effect of different heat stroke time effect on cell permeability of.1.3. according to different heat attack time HUVECs divided into control group, 0.5h group, 1H group, 1.5h group and 2H group by Western Blot method for detection of the same heat blow temperature (43 DEG C) VE-Cadherin total protein HUVECs, content and distribution of.2.p38 MAPK pathway of membrane protein and plasma protein induced endothelial cell permeability changes in the role of 2.1. to construct HUVECs heat stroke model in thermal shock, according to different heat stroke time divided into control Group, 0.5h group, 1H group, 1.5h group and 2H group, p38 Western Blot HUVECs method for the detection of protein phosphorylation in 2.2.; construction of HUVECs heat stroke model, according to the different treatment were divided into control group (SB group, p38 inhibitor SB203580, concentration of 10uM, HS group) and SB+HS (heat stroke group) group. The SB group is in the pre HUVECs joined the SB203580 and in the cell culture box the incubating for 2h, SB+HS Pa in HUVECs group is calculated with SB203580 co incubated with 2h after the 43 hot against 2h. was detected by TEER, HUVECs 2.3., said this permeability; according to different processing factors to HUVECs were divided into control group, SB group, HS group and SB+HS group. Group after treatment by Western blot and immunofluorescence detection of VE-Cadherin in the cell membrane, content and distribution in the cytoplasm. Results: 1. hot blow caused by endothelial cell permeability changes and molecular mechanism of 1.1. against different heat temperature permeability of HUVECs Change: heat hit HUVECs TEER decreased significantly, Pa increased significantly, cell permeability increased. With the heat blow temperature increased, TEER decreased gradually, Pa increased gradually; 1.2. HUVECs under different heat attack time: the permeability change with the heat hit time prolonged, TEER gradually decreased, Pa gradually increased significantly, increased permeability of.1.3. cells hit HUVECs VE-Cadherin thermal change: 1.3.1. in heat stroke process, the amount of VE-Cadherin protein in HUVECs with heat stroke time had no significant changes in the thermal process; 1.3.2., HUVECs VE-Cadherin in the cell membrane was significantly reduced in the cytoplasm significantly increased the content of.2.p38 MAPK pathway to induce endothelial cell permeability changes the role of 2.1. in the early stage of heat stroke can make the phosphorylation level of p38 HUVECs was significantly higher in heat shock; 2.2.p38 inhibitor of MAPK SB203580 pretreatment, heat shock in HU VECs TEER was significantly decreased, Pa was significantly reduced, the stability of the cell permeability; pretreatment with 2.3.SB203580, Western Blot and immunofluorescence indicated that heat stress induced by HUVECs in the VE-Cadherin of the original "membrane reduced, re distribution trend of cytoplasm increased" significantly reduced; the heat shock temperature is a time dependent permeability of HUVECs the VE-Cadherin increased with the heat again blow distribution from the plasma membrane to the cytoplasm. Early heat stroke induced P38 phosphorylation, and through the p38 MAPK pathway of VE-Cadherin from cell membrane to cytoplasm translocation, causing cell adhesion damage caused by the change of permeability of HUVECs.

【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R594.12

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