本文選題：支氣管上皮屏障的破壞胞外Hsp90α + RhoA/MLC信號��； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景支氣管哮喘(簡稱哮喘)是多種細胞成分和細胞組分參與的持續(xù)的慢性氣道炎癥性疾病。氣道屏障的破壞和高通透性是哮喘發(fā)病的啟動環(huán)節(jié)。既往研究表明屋塵螨(HDM)能增加氣道上皮屏障的通透性,從而參與哮喘的發(fā)生發(fā)展。然而,其具體機制尚不明確。熱休克蛋白(Hsp)90α通過分泌到細胞外的微環(huán)境(簡稱胞外)而發(fā)揮其促細胞動力作用,目前的研究主要集中在傷口的愈合和腫瘤的轉(zhuǎn)移。RhoA信號是生物體內(nèi)重要的信號,參與了血管內(nèi)皮及腸道的屏障的破壞,抑制Hsp90可通過破壞src調(diào)節(jié)的RhoA信號保護內(nèi)毒素(LPS)誘導(dǎo)的肺內(nèi)皮細胞的屏障破壞。因此,胞外的Hsp90α(eHsp90α)可能在HDM誘導(dǎo)的氣道上皮屏障破壞中扮演十分重要的作用,同樣,體外研究也進一步表明了抑制表皮生長因子受體信號(EGFR信號)可明顯改善HDM所導(dǎo)致的氣道上皮高通透性,但其具體機制尚不明確。實驗?zāi)康?1、探討HDM誘導(dǎo)的氣道上皮屏障破壞的具體機制和eHsp90α通過激活RhoA/肌球蛋白輕鏈(MLC)介導(dǎo)HDM誘導(dǎo)的支氣管上皮屏障的破壞。2、探討HDM對肌動蛋白應(yīng)力纖維(F-actin)重新排布的影響及EGFR信號在其中的作用。實驗方法:通過測量跨膜電阻值(TEER)和右旋糖苷的通透率(FITC-DX)來評估HDM誘導(dǎo)的人支氣管氣道上皮細胞16HBE14o-(16HBE)的屏障功能的破壞,通過免疫蛋白印記法(Western blotting)和免疫熒光(Cofocal)來評估粘附連接蛋白E-cadherin及β-catenin的表達和分布的情況。通過濃縮和純化條件培養(yǎng)基檢測氣道上皮細胞對eHsp90α的分泌情況。RhoA的活性是通過Rho G-LISA(?)RhoA activation assay kitTM biochem kit試劑盒測定,通過蛋白免疫印記法測定MLC磷酸化水平。另外,應(yīng)用Western blotting方法檢測HDM對EGFR、磷酸化EGFR及F-actin蛋白水平變化,應(yīng)用免疫熒光技術(shù)觀察F-actin的分布變化。實驗結(jié)果:與對照組相比,HDM刺激16HBE細胞引起上皮細胞單層的高通透性,表現(xiàn)為TEER值下降和FITC-DX升高,且均在400U/ml的HDM最明顯。同時,HDM亦可引起粘附連接蛋白E-cadherin和β-catenin的在細胞膜上面的分布異常,由細胞膜向胞漿彌散。另外,HDM刺激16HBE細胞時,可以觀察到eHsp90α的分泌是明顯增多,且均在400 U/ml的HDM最明顯。進一步通過慢病毒下調(diào)Hsp90α及預(yù)處理eHsp90α的單克隆抗體1G6-D7可以保護HDM誘導(dǎo)的屏障的破壞,表現(xiàn)為TEER和FITC的改善及E-cadherin和β-catenin的分布異常的改善。同時,400 U/ml的HDM作用16HBE細胞在6h引起RhoA的活性的增加,而同濃度的HDM可引起MLC的磷酸化水平開始6h升高,24 h達最高。然而發(fā)現(xiàn)下調(diào)Hsp90α及預(yù)處理eHsp90α的單克隆抗體1G6-D7可以保護HDM誘導(dǎo)的RhoA的活性和MLC的磷酸化水平。進一步使用Rho激酶的抑制劑GSK429286A和Y27632 2HC1預(yù)處理16HBE細胞可以保護HDM誘導(dǎo)的支氣管上皮屏障的破壞,表現(xiàn)為TEER和FITC的改善及E-cadherin和β-catenin的分布異常的改善。另外,人重組(hr)Hsp90α而不是hrHsp90β刺激16HBE細胞亦可以誘導(dǎo)支氣管上皮屏障的高通透性及RhoA/MLC信號的活化。然后,予EGFR抑制劑AG-1478進行預(yù)處理,實驗分為四組:對照組,AG-1478組,HDM組,AG-1478+HDM組。與對照組比較,HDM組磷酸化EGFR表達明顯增多,加入EGFR抑制劑AG-1478后E-cadherin和β-catenin的分布異常及TEER值、FITC-DX通透率均明顯改善。HDM刺激后促進F-actin表達增多并出現(xiàn)重新排布,而EGFR抑制劑AG-1478可明顯抑制這一過程。實驗結(jié)論:1、研究證明了 eHsp90α通過激活RhoA/MLC信號參與HDM導(dǎo)致的支氣管上皮屏障的破壞,提示eHsp90α是哮喘管理治療的新靶點。2、EGFR信號通過調(diào)節(jié)F-actin的重新排布促進HDM所導(dǎo)致的氣道上皮屏障破壞。
[Abstract]:Background bronchial asthma (asthma) is a persistent chronic airway inflammatory disease involving multiple cell components and cell components. The destruction of the airway barrier and high permeability are the starting links of asthma. Previous studies have shown that HDM can increase the permeability of the airway epithelial barrier and participate in the development of asthma. However, its specific mechanism is not clear. The heat shock protein (Hsp) 90 alpha plays its cellular power by secreting the extracellular microenvironment (extracellular). The current research focuses on the healing of the wound and the metastasis of the tumor. The.RhoA signal is an important signal number in the organism, and is involved in the destruction of the barrier of the vascular endothelial and intestinal tract. Hsp90 can protect the barrier of endothelial cells induced by endotoxin (LPS) by destroying the RhoA signal regulated by Src. Therefore, the extracellular Hsp90 alpha (eHsp90 alpha) may play a very important role in the destruction of the airway epithelial barrier induced by HDM. In addition, in vitro studies have also demonstrated the inhibition of the epidermal growth factor receptor signal (EGFR letter). It can obviously improve the permeability of the airway epithelium caused by HDM, but its specific mechanism is not clear. 1, the specific mechanism of HDM induced airway barrier destruction and the destruction of.2 by the activation of the RhoA/ myosin light chain (MLC) mediated HDM induced upper barrier of the bronchial skin to the actin stress fiber of HDM The effect of the rearrangement of vitamin (F-actin) and the role of EGFR signal in it. Experimental methods: by measuring the transmembrane resistance value (TEER) and the permeability of dextran (FITC-DX) to evaluate the disruption of the barrier function of 16HBE14o- (16HBE) in human bronchial airway epithelial cells induced by HDM, through the immunoglobulin imprinting (Western blotting) and immunofluorescence (Cofocal) to evaluate the expression and distribution of adhesion connexin E-cadherin and beta -catenin. The activity of.RhoA in the secretion of eHsp90 alpha in airway epithelial cells by concentration and purification condition medium was measured by Rho G-LISA (?) RhoA activation assay kitTM Biochem assay kit and determined by protein immuno imprinting method. C phosphorylation level. In addition, the Western blotting method was used to detect the changes of HDM to EGFR, phosphorylated EGFR and F-actin protein, and the distribution of F-actin was observed by immunofluorescence. Experimental results: compared with the control group, HDM stimulates 16HBE cells to cause the high permeability of the epithelial cell monolayer, showing a decline in TEER and the increase of FITC-DX. HDM is the most obvious in 400U/ml. At the same time, HDM can also cause the abnormal distribution of adhesion connexin E-cadherin and beta -catenin on the cell membrane, diffuse from the cell membrane to the cytoplasm. In addition, when HDM stimulates 16HBE cells, it is observed that the secretion of eHsp90 A is significantly increased, and the 400 U/ml HDM is most obvious. Further down the lentivirus The monoclonal antibody 1G6-D7 of Hsp90 alpha and pretreated eHsp90 alpha can protect the destruction of HDM induced barriers, showing the improvement of TEER and FITC and the improvement of the distribution of E-cadherin and beta -catenin. At the same time, the activity of 16HBE cells in the 400 U/ml HDM action is increased in 6h, and the same concentration may cause the level of phosphorylation. Increase, 24 h reached the highest. However, the monoclonal antibody 1G6-D7 to reduce Hsp90 alpha and pretreat eHsp90 alpha could protect the activity of HDM induced RhoA and the phosphorylation level of MLC. Further use of the inhibitor of Rho kinase, GSK429286A and Y27632 2HC1, can protect the damage of the bronchial epithelial barrier induced by the inducible bronchial epithelium. The improvement of R and FITC and the improvement of the abnormal distribution of E-cadherin and beta -catenin. In addition, human recombinant (HR) Hsp90 alpha rather than hrHsp90 beta stimulation of 16HBE cells could also induce the permeability of the bronchial epithelial barrier and the activation of RhoA/MLC signal. Then, the EGFR inhibitor AG-1478 was pretreated. The experiment was divided into four groups: the control group, the AG-1478 group, and the control group. Group AG-1478+HDM. Compared with the control group, the expression of phosphorylated EGFR in the HDM group increased obviously, and the distribution of E-cadherin and beta -catenin and TEER value after the addition of EGFR inhibitor AG-1478, the FITC-DX penetration rate obviously improved the F-actin expression and rearrangement after.HDM stimulation, and EGFR inhibitor could obviously inhibit this process. Conclusions: 1, the study shows that eHsp90 alpha participates in the disruption of the bronchial epithelial barrier caused by HDM by activating the RhoA/MLC signal, suggesting that eHsp90 alpha is a new target for the management of asthma,.2, and EGFR signal can promote the airway epithelial barrier caused by HDM by regulating the rearrangement of F-actin.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R562.25

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相關(guān)期刊論文 前1條

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本文編號：1794562