本文選題：熱打擊　切入點(diǎn)：絲裂原活化蛋白激酶　出處：《廣州中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：中暑(heat stroke)由于全身炎癥反應(yīng)(SIRS)作用,廣泛存在著血管內(nèi)皮(VEC)損傷,伴隨血管滲漏、出血、組織水腫等病理改變,導(dǎo)致微循環(huán)障礙、休克與多器官功能障礙綜合征(MODS)。血管通透性的改變與血管內(nèi)皮的粘附連接(AJ)及血管內(nèi)皮鈣粘素(VE-Cadherin)的功能結(jié)構(gòu)緊密相關(guān)。然而,中暑的血管通透性變化規(guī)律及其深入分子信號(hào)通路機(jī)制的研究仍有待開展。目的:1.研究熱打擊下人臍靜脈內(nèi)皮細(xì)胞(HUVECs)的通透性變化規(guī)律。2.研究p38 MAPK通路對(duì)熱打擊所致HUVECs通透性變化及對(duì)VE-Cadherin分布變化的影響。方法:1.熱打擊引起內(nèi)皮細(xì)胞通透性變化及其分子機(jī)制1.1.構(gòu)建體外HUVECs熱打擊模型,根據(jù)不同熱打擊溫度分為對(duì)照組、39℃組、41℃組和43℃組,利用Transwell技術(shù)檢測(cè)單層內(nèi)皮細(xì)胞跨膜電阻值(TEER),利用熒光蛋白跨膜滲漏實(shí)驗(yàn)計(jì)算細(xì)胞通透系數(shù)(Pa)。TEER越低、Pa越大,細(xì)胞通透性越高;1.2.根據(jù)不同熱打擊時(shí)間把HUVECs分為對(duì)照組、1h組、2h組、和3h組,檢測(cè)TEER,計(jì)算Pa,研究不同熱打擊時(shí)間對(duì)細(xì)胞通透性的影響。1.3.根據(jù)不同熱打擊時(shí)間把HUVECs分為對(duì)照組、0.5h組、1h組、1.5h組和2h組,利用Western Blot方法檢測(cè)同一熱打擊溫度下(43℃)HUVECs中VE-Cadherin總蛋白、膜蛋白和漿蛋白的含量與分布。2.p38 MAPK通路在熱打擊引起內(nèi)皮細(xì)胞透通性變化中的作用2.1.構(gòu)建HUVECs熱打擊模型,根據(jù)不同熱打擊時(shí)間分為對(duì)照組、0.5h組、1h組、1.5h組和2h組,Western Blot方法檢測(cè)HUVECs中p38蛋白的磷酸化水平;2.2.構(gòu)建HUVECs熱打擊模型,根據(jù)不同的處理因素分為對(duì)照組、SB組(p38抑制劑SB203580,濃度10uM)、HS組(熱打擊組)和SB+HS組。其中,SB組是預(yù)先在HUVECs加入SB203580并于細(xì)胞培養(yǎng)箱中共孵育2h,SB+HS組是在HUVECs加SB203580共孵育2h后行43℃熱打擊2h。檢測(cè)各組TEER,計(jì)算Pa,以此表示HUVECs通透性;2.3.根據(jù)不同處理因素把HUVECs分為對(duì)照組、SB組、HS組和SB+HS組。分組處理后,通過Western blot及免疫熒光的方法檢測(cè)VE-Cadherin在胞膜、胞漿中的含量及分布情況。結(jié)果:1.熱打擊引起內(nèi)皮細(xì)胞透通性變化及其分子機(jī)制1.1.不同熱打擊溫度下HUVECs的通透性改變:熱打擊下HUVECs的TEER顯著降低、Pa顯著增大,細(xì)胞通透性增高。隨熱打擊溫度升高,TEER逐漸降低、Pa逐漸增大;1.2.不同熱打擊時(shí)間下HUVECs的通透性改變:隨熱打擊時(shí)間延長(zhǎng),TEER逐漸顯著降低、Pa逐漸顯著增大,細(xì)胞通透性增高。1.3.熱打擊下 HUVECs 的 VE-Cadherin 改變:1.3.1.在熱打擊過程中,HUVECs中VE-Cadherin蛋白總量隨熱打擊時(shí)間延長(zhǎng)變化無(wú)統(tǒng)計(jì)學(xué)意義;1.3.2.在熱打過程中,HUVECs中VE-Cadherin在胞膜上含量顯著減少,在胞漿中含量顯著增加。2.p38 MAPK通路在熱打擊引起內(nèi)皮細(xì)胞透通性變化中的作用2.1.早期熱打擊即可使HUVECs的p38磷酸化水平顯著增高;2.2.p38 MAPK通路抑制劑SB203580預(yù)處理,熱打擊所致HUVECs的TEER降幅顯著減小、Pa增幅顯著減小,穩(wěn)定了細(xì)胞通透性;2.3.SB203580預(yù)處理,Western Blot及免疫熒光均提示熱打擊引起的HUVECs中VE-Cadherin原"胞膜減少、胞漿增加"的重新分布趨勢(shì)顯著減輕;結(jié)論:熱打擊下HUVECs通透性呈溫度時(shí)間依賴性增高,其VE-Cadherin隨熱打擊發(fā)生從胞膜到胞漿的再分布。熱打擊早期即引起P38磷酸化,并通過p38 MAPK通路調(diào)控VE-Cadherin從胞膜到胞漿的移位,造成細(xì)胞間粘附連接的破壞,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.

【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R594.12

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