胞外的热休克蛋白90α介导屋尘螨导致的支气管上皮屏障破坏过程及其机制的探讨

发布时间：2018-04-24 01:33

本文选题：支气管上皮屏障的破坏胞外Hsp90α + RhoA/MLC信号　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景支气管哮喘(简称哮喘)是多种细胞成分和细胞组分参与的持续的慢性气道炎症性疾病。气道屏障的破坏和高通透性是哮喘发病的启动环节。既往研究表明屋尘螨(HDM)能增加气道上皮屏障的通透性,从而参与哮喘的发生发展。然而,其具体机制尚不明确。热休克蛋白(Hsp)90α通过分泌到细胞外的微环境(简称胞外)而发挥其促细胞动力作用,目前的研究主要集中在伤口的愈合和肿瘤的转移。RhoA信号是生物体内重要的信号,参与了血管内皮及肠道的屏障的破坏,抑制Hsp90可通过破坏src调节的RhoA信号保护内毒素(LPS)诱导的肺内皮细胞的屏障破坏。因此,胞外的Hsp90α(eHsp90α)可能在HDM诱导的气道上皮屏障破坏中扮演十分重要的作用,同样,体外研究也进一步表明了抑制表皮生长因子受体信号(EGFR信号)可明显改善HDM所导致的气道上皮高通透性,但其具体机制尚不明确。实验目的:1、探讨HDM诱导的气道上皮屏障破坏的具体机制和eHsp90α通过激活RhoA/肌球蛋白轻链(MLC)介导HDM诱导的支气管上皮屏障的破坏。2、探讨HDM对肌动蛋白应力纤维(F-actin)重新排布的影响及EGFR信号在其中的作用。实验方法:通过测量跨膜电阻值(TEER)和右旋糖苷的通透率(FITC-DX)来评估HDM诱导的人支气管气道上皮细胞16HBE14o-(16HBE)的屏障功能的破坏,通过免疫蛋白印记法(Western blotting)和免疫荧光(Cofocal)来评估粘附连接蛋白E-cadherin及β-catenin的表达和分布的情况。通过浓缩和纯化条件培养基检测气道上皮细胞对eHsp90α的分泌情况。RhoA的活性是通过Rho G-LISA(?)RhoA activation assay kitTM biochem kit试剂盒测定,通过蛋白免疫印记法测定MLC磷酸化水平。另外,应用Western blotting方法检测HDM对EGFR、磷酸化EGFR及F-actin蛋白水平变化,应用免疫荧光技术观察F-actin的分布变化。实验结果:与对照组相比,HDM刺激16HBE细胞引起上皮细胞单层的高通透性,表现为TEER值下降和FITC-DX升高,且均在400U/ml的HDM最明显。同时,HDM亦可引起粘附连接蛋白E-cadherin和β-catenin的在细胞膜上面的分布异常,由细胞膜向胞浆弥散。另外,HDM刺激16HBE细胞时,可以观察到eHsp90α的分泌是明显增多,且均在400 U/ml的HDM最明显。进一步通过慢病毒下调Hsp90α及预处理eHsp90α的单克隆抗体1G6-D7可以保护HDM诱导的屏障的破坏,表现为TEER和FITC的改善及E-cadherin和β-catenin的分布异常的改善。同时,400 U/ml的HDM作用16HBE细胞在6h引起RhoA的活性的增加,而同浓度的HDM可引起MLC的磷酸化水平开始6h升高,24 h达最高。然而发现下调Hsp90α及预处理eHsp90α的单克隆抗体1G6-D7可以保护HDM诱导的RhoA的活性和MLC的磷酸化水平。进一步使用Rho激酶的抑制剂GSK429286A和Y27632 2HC1预处理16HBE细胞可以保护HDM诱导的支气管上皮屏障的破坏,表现为TEER和FITC的改善及E-cadherin和β-catenin的分布异常的改善。另外,人重组(hr)Hsp90α而不是hrHsp90β刺激16HBE细胞亦可以诱导支气管上皮屏障的高通透性及RhoA/MLC信号的活化。然后,予EGFR抑制剂AG-1478进行预处理,实验分为四组:对照组,AG-1478组,HDM组,AG-1478+HDM组。与对照组比较,HDM组磷酸化EGFR表达明显增多,加入EGFR抑制剂AG-1478后E-cadherin和β-catenin的分布异常及TEER值、FITC-DX通透率均明显改善。HDM刺激后促进F-actin表达增多并出现重新排布,而EGFR抑制剂AG-1478可明显抑制这一过程。实验结论:1、研究证明了 eHsp90α通过激活RhoA/MLC信号参与HDM导致的支气管上皮屏障的破坏,提示eHsp90α是哮喘管理治疗的新靶点。2、EGFR信号通过调节F-actin的重新排布促进HDM所导致的气道上皮屏障破坏。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25

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