整合素-β1在哮喘气道重塑中的作用及shRNA靶向干预研究

发布时间：2018-04-13 19:47

本文选题：哮喘 + ASMC　；参考：《暨南大学》2012年博士论文

【摘要】：气道重塑是支气管哮喘（简称哮喘）的一个重要病理特征，，但其具体发生机制尚不明确。整合素（Integrin）是一种由α、β亚单位组成的二聚体糖蛋白，能通过独特的信号传导途径调控细胞生长、分化、增殖、凋亡等多个生理过程。现已证实，Integrin-β1广泛存在于气道平滑肌细胞（ASMC）表面，并能调控ASMC多个重要的生理过程，故可能对哮喘气喘重塑有重要影响。本研究通过构建靶向沉默小鼠Integrin-β1的shRNA表达载体，从体外、体内水平探讨Integrin-β1在哮喘气道重塑中的作用及其信号调控机制，从而确定哮喘治疗的新靶点。第一部分整合素-β1对哮喘气道重塑的影响目的探讨Integrin-β1对哮喘气道重塑的影响。方法设计并合成靶向小鼠Integrin-β1基因的shRNA表达载体，分别体外、体内转入哮喘小鼠模型ASMC，免疫荧光法检测体内外转染效率，RT-PCR及Real-time PCR法检测体外干预前后Integrin-β1mRNA表达变化；Western-blot法检测体外干预前后Integrin-β1蛋白表达变化；CCK-8法、BrdU ELISA法及流式细胞仪法检测体外干预前后ASMC增殖率及细胞周期分布时相的变化；Annexin V-PE/7-AAD双染法检测体外干预前后ASMC凋亡的变化；Transwell法检测体外干预前后ASMC迁移率的变化；ELISA法检测体外干预前后IL-6、IL-8的含量变化；肺组织病理切片观察体内干预前后哮喘小鼠模型气道重塑变化。结果shRNA表达载体能稳定转染哮喘小鼠ASMC，体外转染后的Integrin-β1的mRNA及其蛋白质水平均显著下调（P均0.05）；细胞增殖率减低（P0.05），G0/G1期细胞比例增加（P0.05），S期细胞比例降低（P0.05）；细胞凋亡率明显升高（P0.05）；此外，细胞迁移速率及IL-6、IL-8的含量则显著降低（P均0.05）；体内转染后的哮喘小鼠模型的气道重塑情况有所减轻。结论Integrin-β1能促使ASMC过度增殖、凋亡减缓、细胞迁移加速、炎症介质IL-6及IL-8分泌增多，引起气道平滑肌层增厚，是导致哮喘气道重塑形成的重要原因。第二部分整合素-β1在哮喘ASMC增殖信号网络中的作用及shRNA靶向干预研究目的探讨Integrin-β1对小鼠慢性哮喘模型ASMC增殖信号网络的调控及shRNA靶向干预作用。方法构建靶向小鼠Integrin-β1基因的shRNA表达载体，体外转入哮喘小鼠模型ASMC，免疫荧光法检测体内外转染效率，RT-PCR及Real-time PCR法检测干预前后CyclinD1、c-Fos的mRNA表达变化；Western-blot法检测干预前后ERK1/2、STAT6、p-ERK1/2、p-STAT6、CyclinD1及c-Fos的蛋白表达变化；CCK-8法、BrdU ELISA法及流式细胞仪法检测干预前后ASMC增殖率及细胞周期时相分布的变化。结果shRNA表达载体稳定转染哮喘小鼠ASMC后，CyclinD1和c-Fos的mRNA及其相应蛋白质水平均显著下调（P均0.05），ERK1/2、STAT6蛋白表达未改变（P均0.05），p-ERK1/2蛋白表达降低（P0.05），而p-STAT6蛋白表达增高（P0.05）；体外转染后的细胞增殖率显著减低（P0.05），细胞周期阻滞在G0/G1时相中（P0.05）。结论哮喘ASMC内Integrin-β1/ERK1/2通路与IL-4/IL-13/STAT6通路间存在交互应答，而Integrin-β1能提高这两条通路的胞核内靶分子CyclinD1及c-Fos活性，可能是ASMC增殖信号网络的“分子开关”及影响哮喘气道重塑的作用机制所在；shRNA表达载体能靶向抑制Integrin-β1基因表达，可望成为哮喘气道重塑基因治疗的有效手段。
[Abstract]:The airway remodeling of bronchial asthma (asthma) is one of the important pathological features, but its pathogenesis is still unclear. Integrin (Integrin) is an alpha, two dimer glycoprotein beta subunit composition, through the unique signal transduction pathways regulating cell growth, differentiation, proliferation, apoptosis and many other the physiological process. It has been confirmed that Integrin- beta 1 exists in airway smooth muscle cells (ASMC) surface, and can regulate the ASMC of a number of important physiological processes, it may have an important impact on asthma asthma remodeling. This study builds the target expression vector to silence shRNA mice Integrin- beta 1 from in vitro, in vivo levels of Integrin- beta 1 in airway remodeling in asthma and its signal regulation mechanism, so as to determine a new target to treat asthma.
The effect of integrin - beta 1 on airway remodeling in asthma
Objective to investigate the effect of Integrin- beta 1 on airway remodeling in asthma.
Methods we designed and synthesized the target expression vector to mouse Integrin- beta 1 gene shRNA respectively in vitro, in vivo into a mouse model of asthma ASMC detection in vivo transfection efficiency of immunofluorescence, expression of Integrin- beta 1mRNA detected before and after the intervention in vitro RT-PCR and Real-time PCR; expression of Integrin- beta 1 protein were detected before and after in vitro Western-blot assay; CCK-8 BrdU method, ELISA method and flow cytometry before and after intervention in vitro detection method ASMC proliferation rate and cell cycle distribution of the phase change; Annexin V-PE/7-AAD double staining changes of ASMC apoptosis assay in vitro before and after intervention; changes of ASMC migration rate before and after intervention by Transwell in vitro; in vitro by ELISA IL-6 detected before and after the intervention, the content of IL-8; changes of airway remodeling in asthmatic mice lung tissue sections were observed before and after intervention.
Results shRNA expression vector can stably transfected asthmatic mice ASMC in vitro after transfection of Integrin- beta 1 and mRNA protein levels were significantly decreased (P < 0.05); reduce the rate of cell proliferation (P0.05), the proportion of cells in G0/G1 phase increased (P0.05), the proportion of cells in S phase decreased (P0.05); the apoptosis rate increased significantly (P0.05); in addition, cell migration rate and IL-6, the content of IL-8 decreased significantly (P < 0.05); airway remodeling in asthmatic mice model in vivo after some relief.
Conclusion Integrin- beta 1 can induce excessive proliferation of ASMC, slow down apoptosis, accelerate cell migration, increase secretion of IL-6 and IL-8 in inflammatory mediators, and cause thickening of airway smooth muscle layer, which is the important cause of airway remodeling in asthma.
The role of the second part integrin beta 1 in the ASMC proliferation signal network of asthma and the study of shRNA targeting intervention
Objective to investigate the effect of Integrin- beta 1 on the regulation of ASMC proliferation signal network and the targeted intervention of shRNA in mice with chronic asthma model.
Methods expression vector to mouse Integrin- beta 1 gene shRNA in vitro to construct target, a mouse model of asthma ASMC detection in vivo transfection efficiency of immunofluorescence, RT-PCR and Real-time were detected before and after intervention by PCR CyclinD1, the expression of c-Fos mRNA detected before and after the intervention of Western-blot; ERK1/2, STAT6, p-ERK1/2, p-STAT6, CyclinD1 and c-Fos expression protein; CCK-8 method, BrdU ELISA method and proliferation rate of ASMC flow cytometry detected before and after the intervention and the distribution of cell cycle changes.
Results shRNA expression vector transfected asthmatic mice after ASMC, CyclinD1 and c-Fos mRNA and their protein levels were significantly lower (P 0.05), ERK1/2, STAT6 protein expression did not change (P < 0.05), lower expression of p-ERK1/2 (P0.05), and the expression of p-STAT6 protein (P0.05); significantly reduce the rate of transfection in vitro after the cell proliferation (P0.05), cell cycle arrest in G0/G1 phase (P0.05).
Conclusion ASMC in asthma exist interactive response Integrin- beta 1/ERK1/2 pathway and IL-4/IL-13/STAT6 pathway, and Integrin- beta 1 can improve the two pathways of target molecules in the nucleus of CyclinD1 and the activity of c-Fos, may be ASMC proliferation signaling networks' molecular switch and the influence mechanism of airway remodeling; shRNA expression vector targeting to inhibit Integrin- beta 1 gene expression, is expected to become an effective means of airway remodeling in asthma gene therapy.

【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R562.25

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