屋尘螨对人支气管上皮细胞通透性和气道平滑肌细胞迁移的影响

发布时间：2018-07-02 19:48

本文选题：支气管哮喘 + 气道重塑　；参考：《南方医科大学》2012年博士论文

【摘要】：研究目的：支气管哮喘是由多种因素导致的慢性气道炎症,虽然现有的药物能够在相当程度上控制哮喘的症状,但却不能有效抑制哮喘的病情进展。气道上皮细胞(AEC)被认为在哮喘的病理生理中处于中心地位,气道上皮损伤、脱落及上皮下纤维化是哮喘气道重塑的重要病理生理特征。而血管内皮生长因子(VEGF)是参与哮喘发病的十分重要的细胞因子,支气管上皮细胞(NHBE)是VEGF的主要来源之一,不同的刺激因素作用下,NHBE对VEGF表达调控状况尚不甚清楚。哮喘患者气道壁网状基底膜中肌纤维母细胞(myofibroblast)数量是增多的,其数量与基底膜的增厚相关。肌纤维母细胞有很强的分泌胶原和胞外基质的能力,其因特征性表达a-平滑肌肌动蛋白(a-SMA)从而具有对环境刺激产生收缩反应的能力,但其细胞来源尚不清楚。屋尘螨(HDM)是诱发和加重哮喘一个重要的致敏因素。本课题利用HDM刺激原代培养的NHBE作为研究对象,与原代培养的人的气道平滑肌细胞共培养,探讨HDM对NHBE分泌VEGF的影响及对气道上皮细胞单层通透性以及上皮下平滑肌细胞迁移的影响,试图寻找治疗哮喘的新的药物靶点。研究内容和方法：1. 人的支气管上皮细胞的原代培养；2. 人的气道平滑肌细胞的原代培养；3. 通过ELISA、荧光定量PCR检测屋尘螨刺激人的支气管上皮细胞后对VEGF表达的影响,通过检测细胞的跨膜电阻了解屋尘螨对人的支气管上皮细胞通透性的影响。4. 采纳共培养装置,荧光显微镜下观察屋尘螨刺激NHBE 对ASMC的趋化作用,酶联免疫吸附法检测培养上清中的细胞因子的水平。5. 统计学方法：采用SPSS13.0统计软件分析,计量资料用均数±标准差(X±S)表示,方差齐时,总体均数的比较采用单向方差分析(one-way ANOVA),各组间多重比较采用LSD法,方差不齐时总体均数的比较采用Welkch法,各组间多重比较采用Tamhane's法检验。以P0.05具有统计学意义。结果：1. 成功培养了可传代达5代的人支气管上皮细胞。2. 成功培养了可传代达8代的人气道平滑肌细胞。3. 不同屋尘螨浓度对NHBE活力的影响：屋尘螨浓度为100 U/ml、200 U/ml、400 U/ml、800 U/ml对 NHBE活力均没有显著影响(P0.05),浓度为1000 U/ml时显著降低细胞活力(P0.001)。4. 屋尘螨可以诱导人的支气管上皮细胞VEGF的表达,且呈明显的时间依赖性和浓度依赖性。在浓度为200U/ml刺激24小时作用最明显,同对照组相比具有显著的统计学意义(P0.05)。同对照组相比,200U/ml尘螨刺激24小时后可明显的增加支气管上皮细胞的通透性,具有显著的统计学意义(P0.05),VEGF的单克隆抗体(贝伐单抗)可以明显阻断这一效应(P0.05)5. 屋尘螨刺激支气管上皮细胞24小时后增加了对气道平滑肌细胞迁移作用,同对照组相比具有显著的统计学意义(P0.05),贝伐单抗可明显阻断这一效应(P0.05)。同步检测transwell下室支气管上皮细胞上清中VEGF浓度,屋尘螨刺激24小时VEGF明显升高,同对照组相比具有显著的统计学意义(P0.05),加用贝伐单抗后明显下降(P0.05)。损伤的支气管上皮细胞上清中检测不到TNF-α、HMGB1相应的增高。结论：1.纤支镜直视下支气管粘膜活检+组织贴块法是一种简便、易行、可重复的人原代支气管上皮细胞的培养方法。2.组织贴块法是一种简便、易行的人原代气道平滑肌细胞的培养方法。3. HD M通过上调VEGF可以直接增加NHBE的通透性。4. HDM通过上调NHBE表达VEGF增加了对气道平滑肌细胞的迁移作用。
[Abstract]:Objective : To study the effect of HDM on the expression of VEGF in asthmatic patients , and to explore the effect of HDM on the expression of VEGF in asthmatic patients . Primary culture of human bronchial epithelial cells ;
2 . Primary culture of human airway smooth muscle cells ;
3 . The influence of house dust mites on the expression of VEGF was detected by ELISA and fluorescence quantitative PCR . The effect of house dust mites on the permeability of human bronchial epithelial cells was investigated by detecting the transmembrane resistance of cells . A co - culture device was adopted to observe the chemoattractant action of NHBE on ASMC under fluorescence microscope , and the level of cytokines in culture supernatant was determined by enzyme linked immunosorbent assay . Statistical methods : SPSS 13.0 was used to analyze the statistical software . The mean per mean difference ( X 卤 S ) of the measurement data was expressed by mean 卤 standard deviation ( X 卤 S ) . One - way ANOVA was used for the comparison of the total mean number between the groups . The comparison between the groups was performed by using the method of LSD . The comparison between the groups was examined by the Tamhane ' s method . The results were as follows : 1 . Human bronchial epithelial cells were successfully cultured for up to 5 generations . Eight generations of human airway smooth muscle cells were successfully cultured . The concentration of house dust mites was 100 U / ml , 200 U / ml , 400 U / ml and 800 U / ml had no significant effect on the activity of NHBE ( P0.05 ) . In comparison with the control group , the effect of 200 U / ml dust mites on the permeability of bronchial epithelial cells was significantly increased ( P0.05 ) . Conclusion : 1 . The tissue labeling method is a simple , easy and reproducible method for culturing human primary bronchial epithelial cells . Conclusion : 1 . The tissue labeling method is a simple , easy and feasible method for culturing human primary bronchial epithelial cells . HD M increases the permeability of NHBE directly by up - regulation of VEGF . HDM increased the migration of vascular smooth muscle cells by up - regulating the expression of VEGF in NHBE .
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R562.25

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