本文選題：炎癥 + 人肺微血管內(nèi)皮細(xì)胞��； 參考：《第三軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：研究背景嚴(yán)重?zé)齻?由于應(yīng)激刺激,神經(jīng)、體液和內(nèi)分泌紊亂,體內(nèi)常有致炎因子釋放,容易引起局部或全身的炎癥反應(yīng)(inflammation)。炎癥反應(yīng)發(fā)生后,炎性細(xì)胞又被進(jìn)一步激活,細(xì)胞和體液均可釋放大量的炎癥介質(zhì),通過(guò)直接損傷血管內(nèi)皮細(xì)胞或間接影響其它化學(xué)因子而最終引起血管通透性的增高。作為人體與外界溝通的重要門(mén)戶,肺在嚴(yán)重?zé)齻?極易受到多種因素影響而引起肺微血管通透性增高,導(dǎo)致肺微血管內(nèi)皮細(xì)胞屏障破壞,進(jìn)而引起急性肺損傷(acute lung injury,ALI)的發(fā)生。截至目前,臨床尚無(wú)有效降低血管通透性的方法,對(duì)于炎癥導(dǎo)致肺微血管通透性增高的機(jī)制我們也依然不清楚。近年來(lái),大量研究結(jié)果提示,微管(microtubule,MT)作為細(xì)胞骨架系統(tǒng)的重要成分,在內(nèi)皮細(xì)胞通透性的調(diào)節(jié)中具有舉足輕重的地位,而微管的穩(wěn)定受到其它多種因素的影響,例如GTP、溫度、藥物、微管相關(guān)蛋白等。相關(guān)研究表明,微管解聚可以引起細(xì)胞形態(tài)、細(xì)胞間連接結(jié)構(gòu)等一系列改變,造成細(xì)胞不可逆的損傷。而微管穩(wěn)定劑則可穩(wěn)定微管,抑制這些破壞作用的發(fā)生。但是在炎癥條件下,微管是如何參與并調(diào)節(jié)人肺微血管內(nèi)皮細(xì)胞(human pulmonary microvascular endothelial cells,HPMECs)通透性的增高,我們尚不清楚。微管相關(guān)蛋白4(microtubule associated protein 4,MAP4)是一種廣泛表達(dá)于全身組織的微管動(dòng)力學(xué)調(diào)節(jié)蛋白,它能促進(jìn)微管聚合,維持細(xì)胞骨架和連接結(jié)構(gòu)的完整。MAP4通過(guò)磷酸化的方式來(lái)調(diào)節(jié)自身活性,當(dāng)其磷酸化增高時(shí),MAP4的活性下降,并且從微管上脫落下來(lái),導(dǎo)致微管動(dòng)力學(xué)的失穩(wěn)。我們前期研究發(fā)現(xiàn),大鼠乳鼠心肌細(xì)胞缺氧后,p38/MAPK(p38 mitogen-activated protein kinase)激活,MAP4磷酸化增高,活性下降,同時(shí)伴隨微管的解聚;此外,我們還發(fā)現(xiàn),燒傷血清處理后,人的臍靜脈內(nèi)皮細(xì)胞(HUVECs)中的p38/MAPK通路激活,并且導(dǎo)致HUVECs通透性的增高。但是,炎癥條件下,HPMECs中MAP4的活性變化情況我們還不清楚,而且MAP4是否參與炎癥導(dǎo)致的HPMECs通透性增高以及可能的調(diào)節(jié)機(jī)制也有待我們進(jìn)一步發(fā)現(xiàn)和證實(shí)。因此,本研究將檢測(cè)map4在炎癥時(shí)的活性變化情況,并且通過(guò)構(gòu)建map4突變體map4(ala),模擬map4的去磷酸化狀態(tài),通過(guò)瞬時(shí)轉(zhuǎn)染重組腺病毒,進(jìn)一步明確map4磷酸化是否介導(dǎo)炎癥引起的hpmecs通透性增高。在此基礎(chǔ)之上,我們還將初步探索map4參與調(diào)節(jié)炎癥導(dǎo)致hpmecs通透性增高的相關(guān)機(jī)制。旨在為臨床相關(guān)疾病的治療提供新的靶點(diǎn)和研究方向。材料和方法:1、我們以hpmecs為研究對(duì)象,利用致炎因子脂多糖(lipopolysaccharide,lps)和腫瘤壞死因子-α(tumournecrosisfactor-α)處理細(xì)胞,模擬體外炎癥刺激。觀察致炎因子lps(200、500、1000ng/ml)和tnf-α(200、500、1000ng/ml)處理前后,hpmecs通透性及微管的變化情況,并采用以下方法進(jìn)行相應(yīng)檢測(cè)和觀察:1)檢測(cè)fitc-dextran(40kda)熒光物質(zhì)漏出情況和測(cè)量跨內(nèi)皮細(xì)胞電阻值(ter),以此觀察肺微血管內(nèi)皮細(xì)胞屏障功能的改變狀況;2)對(duì)α-微管蛋白進(jìn)行免疫熒光染色(immunofluorescence,if),以此觀察微管的形態(tài)、結(jié)構(gòu)和分布狀況;3)將游離態(tài)與聚合態(tài)的微管蛋白分離、提取并定量,采用westernblot(wb)方法進(jìn)行檢測(cè),觀察游離態(tài)與聚合態(tài)微管蛋白含量的變化情況。2、使用微管穩(wěn)定劑紫杉醇(taxol,1um)預(yù)處理細(xì)胞,觀察紫杉醇預(yù)處理前后,致炎因子lps(500ng/ml)和tnf-α(500ng/ml)刺激對(duì)hpmecs通透性和微管的影響。通過(guò)檢測(cè)fitc-dextran和ter,來(lái)觀察肺微血管內(nèi)皮細(xì)胞單層通透性的改變情況;采用if法觀察微管的形態(tài)、結(jié)構(gòu)和分布的改變;分離并提取游離態(tài)和聚合態(tài)的微管蛋白,通過(guò)wb檢測(cè)其含量的變化。3、通過(guò)wb進(jìn)行檢測(cè),觀察致炎因子lps(500ng/ml)和tnf-α(500ng/ml)處理hpmecs1h、3h、6h、12h后,map4活性的變化情況。構(gòu)建模擬map4去磷酸化狀態(tài)的突變體map4(ala),瞬時(shí)轉(zhuǎn)染hpmecs72h,通過(guò)測(cè)量fitc-dextran熒光漏出和ter,觀察轉(zhuǎn)染前后,致炎因子lps(500ng/ml)和tnf-α(500ng/ml)刺激對(duì)hpmecs通透性的影響,并采用if法檢測(cè)微管的形態(tài)、結(jié)構(gòu)和分布狀況,同時(shí)分離提取聚合態(tài)與游離態(tài)的微管蛋白,通過(guò)wb法檢測(cè)其含量的改變,另外,使用免疫共沉淀(immunoprecipitation,ip)檢測(cè)map4與微管間的相互作用情況,以此明確map4在調(diào)節(jié)hpmecs通透性和微管改變中發(fā)揮的作用。4、以wb法檢測(cè)致炎因子lps(500ng/ml)和tnf-α(500ng/ml)處理hpmecs1h、3h、6h、12h后,p38/mapk的激活情況。構(gòu)建p38/mapk的上游激酶mkk6(glu),同時(shí)應(yīng)用p38/mapk抑制劑sb203580(5um),觀察mkk6(glu)轉(zhuǎn)染細(xì)胞和sb203580預(yù)處理細(xì)胞前后,致炎因子lps(500ng/ml)和tnf-α(500ng/ml)刺激對(duì)map4(s696、s787、s768)和p-p38活性的變化情況,同時(shí)采用ip法檢測(cè)map4和微管間的相互作用,以此明確p38/mapk是否為map4的上游激酶。5、應(yīng)用p38/mapk上游激酶mkk6(glu)和p38/mapk抑制劑sb203580(5um),通過(guò)檢測(cè)fitc-dextran和ter,觀察sb203580預(yù)處理細(xì)胞前后,致炎因子lps(500ng/ml)和tnf-α(500ng/ml)刺激或mkk6(glu)轉(zhuǎn)染后對(duì)hpmecs通透性的影響,采用if法檢測(cè)微管的形態(tài)結(jié)構(gòu)和分布狀況,通過(guò)分離提取聚合/游離態(tài)的微管蛋白,使用wb法檢測(cè)其含量改變情況。同時(shí),轉(zhuǎn)染或共轉(zhuǎn)染cmv-null、map4(ala)和mkk6(glu),并采用上述方法檢測(cè)hpmecs通透性、微管結(jié)構(gòu)以及游離/聚合態(tài)微管蛋白含量的變化情況,以明確p38/mapk激酶通路是否通過(guò)調(diào)節(jié)map4活性參與并調(diào)控lps和tnf-α介導(dǎo)的hpmecs通透性與微管的改變。6、利用caspase-3抑制劑(z-dqmd-fmk,10um)、p38/mapk抑制劑sb203580(5um)和map4(ala)預(yù)處理或轉(zhuǎn)染細(xì)胞,觀察預(yù)處理細(xì)胞前后,致炎因子lps(500ng/ml)和tnf-α(500ng/ml)刺激對(duì)hpmecs凋亡和hpmecs通透性的影響,通過(guò)tunel法檢測(cè)細(xì)胞凋亡,測(cè)量fitc-dextran和ter來(lái)評(píng)判hpmecs通透性的改變,以此明確map4介導(dǎo)的炎癥引起的hpmecs通透性增高不依賴細(xì)胞凋亡。結(jié)果1、致炎因子lps(200、500、1000ng/ml)或tnf-α(200、500、1000ng/ml)處理hpmecs6h后,hpmecs單層對(duì)fitc-dextran熒光物質(zhì)漏出增多,跨內(nèi)皮細(xì)胞電阻值降低,并且呈劑量依賴性改變;致炎因子lps或tnf-α刺激濃度為200ng/ml時(shí),微管結(jié)構(gòu)不規(guī)則,少量微管斷裂;lps或tnf-α刺激濃度為500ng/ml時(shí),細(xì)胞膜周微管解聚明顯,細(xì)胞核周微管皺縮;當(dāng)lps或tnf-α刺激濃度增至1000ng/ml時(shí),只殘留極少的微管結(jié)構(gòu)和片段;游離/聚合態(tài)微管蛋白定量檢測(cè)提示,致炎因子lps(200、500、1000ng/ml)或tnf-α(200、500、1000ng/ml)處理后,游離態(tài)微管蛋白增多而聚合態(tài)微管蛋白減少,并呈劑量依賴性改變。2、根據(jù)上述實(shí)驗(yàn)結(jié)果,選擇lps(500ng/ml)或tnf-α(500ng/ml)兩個(gè)典型的致炎因子用于后續(xù)實(shí)驗(yàn)。在微管穩(wěn)定劑紫杉醇(1um)預(yù)處理細(xì)胞1h后,正常細(xì)胞通透性無(wú)明顯改變,微管聚合增加,解聚減少;同時(shí),致炎因子lps(500ng/ml)或tnf-α(500ng/ml)刺激6h導(dǎo)致的fitc-dextran熒光物質(zhì)漏出明顯減少,跨內(nèi)皮細(xì)胞電阻值明顯增加;并且微管網(wǎng)狀結(jié)構(gòu)破壞減少,細(xì)胞膜周微管密度增加;聚合態(tài)微管蛋白含量增加,游離態(tài)微管蛋白含量減少。3、致炎因子lps(500ng/ml)或tnf-α(500ng/ml)處理hpmecs6h后,map4(s696與s787)磷酸化增高,而map4(ser768)磷酸化無(wú)改變。轉(zhuǎn)染map4(ala)干預(yù)map4磷酸化后,正常hpmecs通透性無(wú)明顯改變,微管聚合增加,解聚減少;同時(shí),致炎因子lps(500ng/ml)或tnf-α(500ng/ml)刺激6h后的hpmecs單層對(duì)fitc-dextran熒光物質(zhì)漏出明顯減少,跨內(nèi)皮細(xì)胞電阻值明顯升高;而且微管的動(dòng)力學(xué)更加穩(wěn)定,微管的結(jié)構(gòu)得到改善,游離態(tài)的微管含量減少而聚合態(tài)微管蛋白增多;此外,map4與微管之間的相互結(jié)合也增多。4、致炎因子lps(500ng/ml)或tnf-α(500ng/ml)處理hpmecs1h、3h、6h、12h后,p38/mapk激活,并且呈時(shí)間依賴性改變。同時(shí),轉(zhuǎn)染mkk6(glu)持續(xù)激活p38/mapk后,map4(s696與s787)和p38磷酸化水平顯著增加,map4與微管相互結(jié)合減少;反之,p38/mapk阻斷劑sb203580(5um)預(yù)處理細(xì)胞后,致炎因子lps(500ng/ml)或tnf-α(500ng/ml)刺激6h導(dǎo)致的p38/mapk激活顯著受抑,且map4(s696與s787)磷酸化水平明顯下降,此外,map4與微管的相互結(jié)合也顯著增多。5、sb203580(5um)預(yù)處理1h后,致炎因子lps(500ng/ml)或tnf-α(500ng/ml)刺激的hpmecs單層對(duì)fitc-dextran熒光物質(zhì)漏出明顯減少,跨內(nèi)皮細(xì)胞電阻值明顯增高;且微管結(jié)構(gòu)破壞受抑,游離態(tài)的微管蛋白含量減少而聚合態(tài)的微管蛋白含量增多;相反,mkk6(glu)轉(zhuǎn)染細(xì)胞后,hpmecs單層對(duì)fitc-dextran熒光物質(zhì)漏出增多,跨內(nèi)皮細(xì)胞電阻值降低,微管解聚增加而聚合減少。此外,我們轉(zhuǎn)染mkk6(glu)+cmv-null后發(fā)現(xiàn),hpmecs單層對(duì)fitc-dextran熒光漏出增多,跨內(nèi)皮細(xì)胞電阻值降低;微管結(jié)構(gòu)破壞,游離態(tài)微管蛋白增加而聚合態(tài)微管蛋白減少;而共轉(zhuǎn)染mkk6(glu)和map4(ala)后,上述變化得到明顯抑制。6、caspase-3抑制劑(z-dqmd-fmk,10um)預(yù)處理細(xì)胞1h后,致炎因子lps(500ng/ml)或tnf-α(500ng/ml)誘導(dǎo)的細(xì)胞凋亡明顯受抑;同時(shí),輔以sb203580(5um)預(yù)處理和map4(ala)轉(zhuǎn)染后,致炎因子lps(500ng/ml)或tnf-α(500ng/ml)刺激的hpmecs單層對(duì)fitc-dextran熒光物質(zhì)漏出仍然減少,跨內(nèi)皮細(xì)胞電阻值仍然增加,提示map4磷酸化介導(dǎo)的致炎因子所致hpmecs通透性增加不依賴細(xì)胞凋亡。結(jié)論致炎因子lps或tnf-α處理hpmecs后,細(xì)胞中p38/mapk激酶通路激活,通過(guò)map4(s696與s787)磷酸化使map4失活,并引起微管解聚,導(dǎo)致hpmecs通透性增高;同時(shí),map4磷酸化介導(dǎo)的致炎因子所致的hpmecs通透性增高不依賴細(xì)胞凋亡。以上結(jié)果提示map4介導(dǎo)的微管結(jié)構(gòu)變化可能參與了炎癥導(dǎo)致的hpmecs通透性增高,有助于我們進(jìn)一步深入認(rèn)識(shí)燒傷休克、炎癥時(shí)急性肺損傷的發(fā)病機(jī)理,為臨床相關(guān)疾病的干預(yù)和治療提供新靶點(diǎn)和方向。
[Abstract]:After severe burns, due to stress stimulation, nerve, body fluid and endocrine disorder, the release of inflammatory factors is often caused in the body, and it is easy to cause local or systemic inflammatory response (inflammation). After the inflammation, inflammatory cells are further activated, and cells and body fluid can release a large number of inflammatory mediators and directly damage the blood vessels. Skin cells or indirect effects of other chemical factors lead to increased vascular permeability. As an important portal for communication between the human body and the outside world, lung microvascular permeability increases greatly after severe burns, causing lung microvascular endothelial cell barrier damage and causing acute lung injury (acute lung inj). Ury, ALI). Up to now, there is no effective method for reducing vascular permeability, and we are still not clear about the mechanism of inflammation leading to increased pulmonary microvascular permeability. In recent years, a large number of research results suggest that microtubule (microtubule, MT), as an important component of the cytoskeleton system, can be used in the regulation of endothelial cell permeability. The stability of the microtubule is affected by many other factors, such as GTP, temperature, drug, microtubule related protein and so on. Related studies have shown that microtubule depolymerization can cause a series of changes in cell morphology, intercellular connection structure and other changes, causing cell irreversible damage. Microtubule stabilizers can stabilize microtubules and inhibit these breaks. It is not clear how microtubules participate in and regulate the permeability of human pulmonary microvascular endothelial cells (HPMECs) in human lung microvascular endothelial cells under inflammatory conditions. Microtubule related protein 4 (microtubule associated protein 4, MAP4) is a microtubule that is widely expressed in whole body tissue. The kinetic regulation protein, which promotes microtubule polymerization, maintains a complete.MAP4 of the cytoskeleton and connection structure to regulate its own activity by phosphorylation. When its phosphorylation is increased, the activity of MAP4 is reduced and the microtubule dynamics is unstable. Our previous study found that the rat myocardial cells were deficient in the rat milk. After oxygen, p38/MAPK (p38 mitogen-activated protein kinase) activated, MAP4 phosphorylation increased, activity decreased and microtubule disaggregation. In addition, we also found that after burn serum treatment, the p38/MAPK pathway in human umbilical vein endothelial cells (HUVECs) was activated and led to the increase of HUVECs permeability. But, under inflammatory conditions, MA in HPMECs We are not clear about the changes in the activity of P4, and the increase in HPMECs permeability and the possible regulatory mechanism of MAP4 in inflammation need further discovery and confirmation. Therefore, this study will detect the changes in the activity of Map4 in inflammation, and simulate dephosphorylation of Map4 by constructing a Map4 mutant Map4 (ALA). State, through transient transfection of recombinant adenovirus, further clarify whether Map4 phosphorylation mediated inflammation induced hpmecs permeability increase. On the basis of this, we will also initially explore the relevant mechanisms of Map4 to regulate the increase of hpmecs permeability caused by inflammation. The aim is to provide new targets and research directions for the treatment of clinical related diseases. And methods: 1, we use hpmecs as the research object, using lipopolysaccharide (LPS) and tumor necrosis factor - alpha (tumournecrosisfactor- alpha) to treat the cells in vitro, and simulate the inflammatory stimulation in vitro. The changes of hpmecs permeability and microtubule changes are observed before and after the treatment of LPS (2005001000ng/ml) and tnf- alpha (2005001000ng/ml). The following methods were used to detect and observe the following methods: 1) detect the leakage of FITC-dextran (40kDa) fluorescent substance and measure the resistance value of the cross endothelial cell (TER) to observe the changes in the barrier function of the pulmonary microvascular endothelial cells, and 2) to observe the microtubule by immunofluorescence staining (immunofluorescence, if) on the alpha microtubulin. State, structure and distribution status; 3) separation of free and polymerized microtubules, extracted and quantified, detected by Westernblot (WB), observed the changes in free and polymerized microtubule protein content.2, using microtubule stabilizer paclitaxel (Taxol, 1um) pretreated cells, before and after paclitaxel pretreatment, to observe the inflammatory factor LPS (50). 0ng/ml) and the effect of tnf- alpha (500ng/ml) stimulation on the permeability and microtubule of hpmecs. By detecting FITC-dextran and ter, the changes in the permeability of the pulmonary microvascular endothelial cells were observed. The morphology, structure and distribution of microtubules were observed by if, and the microtubules in the isolated and polymerized states were isolated and extracted, and the content of the microtubules was detected by WB. .3, detected by WB, and observed the changes in the activity of hpmecs1h, 3h, 6h, 12h after hpmecs1h, 3h, 6h, 12h, and tnf- alpha (500ng/ml) in treating hpmecs1h, 3h, 6h, 12h. The effect of ML) and tnf- alpha (500ng/ml) stimulation on the permeability of hpmecs, and using if to detect the morphology, structure and distribution of microtubules, and the separation and extraction of microtubules of the polymeric and free microtubules, and the changes in the content of the microtubules by WB method. In addition, the interaction between Map4 and microtubules was detected by the immunoprecipitation (immunoprecipitation, IP). In order to clarify the role of Map4 in regulating hpmecs permeability and microtubule change,.4 was used to detect the activation of hpmecs1h, 3h, 6h, 12h, and tnf- alpha (500ng/ml) were detected by WB method. The changes in the activity of Map4 (s696, s787, s768) and p-p38 were induced by LPS (500ng/ml) and tnf- alpha (500ng/ml) before and after the pretreatment of SB203580 cells, and the interaction between Map4 and microtubules was detected by IP method. 203580 (5um), by detecting FITC-dextran and ter, the effects of LPS (500ng/ml) and tnf- alpha (500ng/ml) stimulation or MKK6 (Glu) on the hpmecs permeability were observed before and after SB203580 pretreatment cells. The morphology and distribution of microtubules were detected by if method, and microtubules were extracted by separation and dissociation. At the same time, cmv-null, Map4 (ALA) and MKK6 (Glu) were transfected or co transfected, and the changes of hpmecs permeability, microtubule structure and free / polymerized microtubule protein content were detected by the above methods to determine whether the p38/mapk kinase pathway was involved in the regulation of Map4 activity and regulated the hpmecs permeability mediated by LPS and tnf- alpha. .6, caspase-3 inhibitor (z-dqmd-fmk, 10um), p38/mapk inhibitor SB203580 (5um) and Map4 (ALA) were pretreated or transfected to cells. The effects of inflammatory factors LPS (500ng/ml) and tnf- alpha stimulation on apoptosis and permeability were observed before and after the pretreated cells. Ran and ter were used to judge the changes in the permeability of hpmecs so that the increase of hpmecs permeability caused by Map4 mediated inflammation was not dependent on apoptosis. Results 1, after LPS (2005001000ng/ml) or tnf- alpha (2005001000ng/ml) treated hpmecs6h, hpmecs monolayer increased the leakage of FITC-dextran fluorescein, and the resistance of trans endothelial cells decreased. When the concentration of LPS or tnf- alpha was 200ng/ml, the microtubule structure was irregular and a small number of microtubules were broken. When the concentration of LPS or tnf- alpha was 500ng/ml, the cell membrane microtubules were depolymerization obviously and the peripheral microtubules were crinkled. When the concentration of LPS or tnf- alpha was increased to 1000ng/ml, only a few microtubule structures remained. Fragments; free / polymerized microtubule protein quantitative detection suggested that after the treatment of inflammatory factor LPS (2005001000ng/ml) or tnf- alpha (2005001000ng/ml), free microtubule protein was increased and the polymerized microtubule decreased, and.2 was changed in a dose-dependent manner. According to the experimental results, two typical inflammation of LPS (500ng/ml) or tnf- alpha (500ng/ml) was selected. After the pretreated cell 1H by microtubule stabilizer paclitaxel (1um), the permeability of normal cells was not obviously changed, microtubule polymerization increased and the depolymerization decreased; meanwhile, the leakage of FITC-dextran fluorescent substance caused by LPS (500ng/ml) or tnf- alpha (500ng/ml) stimulated 6h significantly decreased, and the resistance value of trans endothelial cells increased significantly. The microtubule microtubule density of microtubule was decreased and the density of microtubule microtubules increased, the content of microtubule protein in polymerized microtubule increased, the content of free microtubule protein decreased by.3, and the phosphorylation of Map4 (s696 and s787) increased, while Map4 (ser768) phosphorylation was not changed after hpmecs6h of LPS (500ng/ml) or tnf- alpha (500ng/ml). After that, there was no obvious change in the permeability of the normal hpmecs, the increase of microtubule polymerization and the decrease of the depolymerization. At the same time, the leakage of the fluorescent substance in the hpmecs monolayer after the stimulation of the inflammatory factor LPS (500ng/ml) or tnf- a (500ng/ml) was significantly reduced, the resistance value of the trans endothelial cells increased obviously, and the microtubule dynamics was more stable and the microstructure of microtubules was improved. The free microtubule content was reduced and the aggregate microtubule increased; in addition, the combination of Map4 and microtubule increased by.4, and inflammatory factor LPS (500ng/ml) or tnf- alpha (500ng/ml) treated hpmecs1h, 3h, 6h, 12h after hpmecs1h, 3h, 6h, 12h, and showed a time dependent change. The level of 38 phosphorylation increased significantly, and the combination of Map4 and microtubule decreased. On the contrary, p38/mapk blocker SB203580 (5um) pretreated the cells, and the activation of p38/mapk activation caused by LPS (500ng/ml) or tnf- alpha (500ng/ml) stimulated 6h, and Map4 (s696 and 500ng/ml) phosphorylation level decreased significantly. Furthermore, the combination of microtubules and microtubules was also significant. After the increase of.5, SB203580 (5um) pretreated 1H, the leakage of hpmecs monolayer stimulated by LPS (500ng/ml) or tnf- alpha (500ng/ml) significantly decreased the leakage of FITC-dextran fluorescent substance and increased the resistance value of the trans endothelial cells, and the microtubule structure destruction was suppressed, the free microtubule content was reduced and the microtubule protein content in the polymerized state increased; on the contrary, MKK After transfection of 6 (Glu) cells, the leakage of FITC-dextran fluorescent substance increased in hpmecs monolayer, the resistance value of cross endothelial cells decreased, microtubule depolymerization increased and polymerization decreased. Furthermore, after transfecting MKK6 (Glu) +cmv-null, we found that the hpmecs monolayer increased the fluorescent leakage of FITC-dextran, reduced the resistance value of the endothelial cells, microtubule structure destruction and free microtubules. The protein increased and the polymerized microtubule decreased, and after CO transfection of MKK6 (Glu) and Map4 (ALA), the above changes were obviously inhibited by.6. After the caspase-3 inhibitor (z-dqmd-fmk, 10um) pretreated the cell 1H, the apoptotic cell apoptosis induced by LPS (500ng/ml) or tnf- alpha was obviously suppressed. After staining, the leakage of hpmecs monolayer stimulated by inflammatory factor LPS (500ng/ml) or tnf- alpha (500ng/ml) still decreases, and the resistance value of trans endothelial cells still increases. It suggests that the increase of hpmecs permeability induced by Map4 phosphorylation mediated inflammation factor is not dependent on cell withering. Conclusion the inflammation factor LPS or tnf- alpha treatment hpmecs, cells P38/mapk kinase pathway is activated by phosphorylation of Map4 (s696 and s787) to deactivate Map4 and cause microtubule depolymerization and lead to higher hpmecs permeability. Meanwhile, the increase of hpmecs permeability induced by Map4 phosphorylation mediated inflammation factor is not dependent on apoptosis. The above results suggest that Map4 mediated microtubule structure changes may be involved in inflammation LED HPME. The increase of CS permeability will help us to further understand the pathogenesis of acute lung injury in burn shock and inflammation, and provide new targets and directions for the intervention and treatment of clinical related diseases.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R54

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