染色质重塑因子Brg1通过E-cadherin影响哮喘气道上皮完整性的研究

发布时间：2017-12-28 06:14

本文关键词：染色质重塑因子Brg1通过E-cadherin影响哮喘气道上皮完整性的研究　出处：《重庆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：第一部分 Brg1在哮喘C57BL/6小鼠的肺组织和气道上皮异常表达目的:观察染色质重塑因子Brg1基因在哮喘C57BL/6小鼠的肺组织和气道上皮的异常表达,了解Brg1和哮喘的相关性。方法:将小鼠分为两组:正常对照组(control)和哮喘组(asthma)。小鼠在最后一次雾化48 h内依次进行AHR检测、支气管肺泡灌洗和BALF细胞计数,同时取BALF细胞沉渣涂片用于细胞分类计数,取肺病理组织用于HE染色、E-cadherin免疫组化染色,取肺组织标本用于提m RNA和蛋白进行Q-PCR和WB检测。结果:肺组织中Brg1的m RNA和蛋白水平在C57BL/6小鼠哮喘组明显升高,抗Brg1免疫组化染色显示Brg1在哮喘组的气道上皮上表达增多。结论:哮喘中Brg1的m RNA和蛋白水平均有明显升高,提示Brg1和哮喘有一定的相关性。同时发现Brg1在哮喘组的气道上皮上高表达,提示Brg1与气道上皮可能有一定的关系,进一步对Brg1进行研究也许可以发现哮喘气道上皮损伤后修复的其他机制。第二部分Brg1基因敲除对C57BL/6哮喘小鼠气道上皮完整性的影响目的:观察Brg1基因对哮喘小鼠AHR、气道炎症和上皮marker E-cadherin的影响。方法:将Cre重组酶转基因小鼠(SFTPC-rt TA/(Tet O)7)与lox P转基因杂合子小鼠(Brg1lox P/lox P)杂交后筛出纯合型Brg1敲除鼠(Brg1~(-/-))。将哮喘小鼠分为四组:野生型小鼠正常对照组(WT ctrl),野生型小鼠哮喘组(WT asthma),Brg1基因敲除鼠正常对照组(Brg1~(-/-)ctrl),Brg1基因敲除鼠哮喘组(Brg1~(-/-)asthma)。小鼠在最后一次雾化48 h内依次进行有创肺功能气道高反应性AHR检测、支气管肺泡灌洗和BALF细胞计数,同时,BALF细胞沉渣涂片用于细胞分类计数,肺病理组织用于HE染色、E-cadherin免疫组化染色,取肺组织标本用于提m RNA和蛋白进行荧光定量PCR和WB检测。结果:有创肺功能AHR检测、BALF细胞计数结果显示,与WT asthma组相比,Brg1~(-/-)asthma组的AHR明显下降,BALF中气道炎症细胞和嗜酸性细胞的数量均有明显降低。HE染色明确显示Brg1~(-/-)asthma组气道周围炎症细胞的浸润明显减轻,气道上皮的完整性得到一定程度的改善。抗上皮marker E-cadherin免疫组化染色显示与WT asthma组的E-cadherin低表达相比,E-cadherin在Brg1~(-/-)asthma组的气道上皮中表达增高,而Brg1~(-/-)Ctrl组的E-cadherin的表达与WT Ctrl组相比无明显差异。气道炎症细胞、嗜酸性细胞数量与AHR指标LR均呈相关性。结论:Brg1基因的敲除可以明显降低C57BL/6哮喘小鼠的AHR、BALF中气道炎症细胞和嗜酸性粒细胞的数量和气道周围炎症细胞的浸润,同时增强了气道上皮marker E-cadherin的表达,提示Brg1基因的敲除可以增强哮喘气道上皮的完整性。第三部分 Brg1基因敲低对人支气管上皮细胞16HBE的修复能力的机制研究目的:利用慢病毒Brg1-sh RNA转染人支气管上皮细胞16HBE敲低Brg1后,观察Brg1对细胞增殖和迁移能力的影响及机制研究。方法:构建慢病毒Brg1干扰载体后,用构建的慢病毒表达载体和包装质粒(packaging mix)共转染293T细胞,包装病毒,用包装好的慢病毒(Brg1-sh RNA)以及慢病毒阴性对照(Brg1-sc RNA)感染16HBE,通过Western Blot和q PCR方法验证后得到稳定株。分别用细胞划痕实验、Transwell小室和CCK8、流式测细胞周期技术检测两组细胞株的迁移和增殖能力。将两种细胞株进行E-cadherin的荧光定量PCR和WB检测,同时荧光定量PCR检测另外两个上皮markers ZO-1和Occlubin1。双重荧光素酶报告基因分析两组细胞Brg1与E-cadherin的启动子区有无相互作用,Ch IP-PCR检测两组细胞Brg1与E-cadherin的3个启动子区序列的结合强度。结果:细胞划痕实验和Transwell小室实验结果显示Brg1基因的敲低可以促进气道上皮细胞的迁移能力,CCK8检测和流式测细胞周期结果显示Brg1基因的敲低可以促进气道上皮细胞的增殖能力,增殖能力增强主要表现为细胞周期的S期增高。荧光定量PCR和WB的结果显示,与未转染的16HBE细胞株和转染Brg1-sc RNA的细胞株相比较,转染Brg1-sh RNA的细胞株E-cadherin的m RNA水平和蛋白表达水平明显增高,而ZO-1和Occlubin1的m RNA水平无明显改变。双重荧光素酶报告基因显示,与转染Brg1-sh RNA的细胞株相比,转染Brg1-sc RNA的细胞株中Brg1与E-cadherin的启动子区的-1964/+941bp有较强结合,而其它序列无明显差异。Ch IP-PCR结果显示Brg1与E-cadherin的启动子区的第1段序列-86/+60 bp在转染Brg1-sc RNA的细胞株中结合最强,而另外两个启动子区序列在两组细胞株中无明显差异。结论:Brg1基因敲低后可以提高16HBE细胞的迁移和增殖能力,主要是Brg1基因通过与E-cadherin启动子区的-86/+60 bp结合来抑制E-cadherin的转录激活。
[Abstract]:The first part is the abnormal expression of Brg1 in the lung tissue and airway epithelium of asthmatic C57BL/6 mice. Objective: To observe the abnormal expression of chromatin remodeling factor Brg1 gene in the lung tissue and airway epithelium of asthmatic C57BL/6 mice, and to understand the correlation between Brg1 and asthma. Methods: the mice were divided into two groups: the normal control group (control) and the asthma group (asthma). The mice in the last aerosol within 48 h by AHR detection, bronchoalveolar lavage and BALF cell count and BALF cells were used for cell counting and classification of sediment smear, the pulmonary pathological tissue for HE staining and immunohistochemical staining of E-cadherin, lung tissue specimens were taken for m RNA and Q-PCR protein and WB detection. Results: the m RNA and protein levels of Brg1 in lung tissue increased significantly in asthmatic group of C57BL/6 mice. Anti Brg1 immunohistochemical staining showed that Brg1 increased in airway epithelium of asthmatic group. Conclusion: the levels of M RNA and protein in Brg1 were significantly increased in asthma, suggesting a certain correlation between Brg1 and asthma. It is also found that Brg1 is highly expressed on airway epithelium of asthma group, suggesting that Brg1 may be related to airway epithelium. Further study of Brg1 may reveal other mechanisms of airway epithelial injury after asthma. The second part is the effect of knockout of Brg1 gene on the integrity of airway epithelium in C57BL/6 asthmatic mice. Objective: To observe the effect of Brg1 gene on AHR, airway inflammation and epithelial marker E-cadherin in asthmatic mice. Methods: Cre transgenic mice (SFTPC-rt TA/ (Tet O) 7) and LOX P (Brg1lox P/lox transgenic mice heterozygous P) after hybridization screening of homozygous Brg1 knockout mice (Brg1~ (- / -)). The asthma mice were divided into four groups: wild type mice in normal control group (WT CTRL), wild type mice asthma group (WT asthma), Brg1 knockout mice in normal control group (Brg1~ (- / -) CTRL), Brg1 gene knockout mouse asthma group (Brg1~ (- / -) asthma). The mice in the last aerosol within 48 h of invasive pulmonary function in airway hyperresponsiveness AHR detection, bronchoalveolar lavage and BALF cell count of BALF cells, at the same time, sediment smear for cell counts, pulmonary pathological tissue for HE staining and immunohistochemical staining of E-cadherin, lung tissue specimens were taken for M and RNA protein quantitative detection of WB and PCR. Results: invasive pulmonary function AHR detection, BALF cell counting results showed that compared with the WT group asthma, Brg1~ (- / -) asthma group AHR decreased significantly, airway inflammatory cells and eosinophil number were reduced obviously in BALF. HE staining clearly shows that Brg1~ (- / -) infiltration of inflammatory cells around the airway in group asthma was significantly reduced, the integrity of the airway epithelium is improved to a certain extent. Anti epithelial marker E-cadherin immunohistochemical staining showed that WT and asthma group compared with the low expression of E-cadherin, E-cadherin in Brg1~ (- / -) expression in airway epithelial asthma group, Ctrl group and Brg1~ (- / -) the expression of E-cadherin and WT in Ctrl group showed no significant difference. The number of airway inflammatory cells and eosinophils was correlated with the AHR index LR. Conclusion: Brg1 gene knockout can significantly reduce the infiltration of C57BL/6 AHR and BALF in asthmatic mice airway inflammatory cells and the number of eosinophils and airway inflammatory cells, and enhanced the expression of marker in airway epithelia of E-cadherin, suggesting that Brg1 gene knockout can enhance the integrity of airway epithelium. The third part is the mechanism of Brg1 knockdown on 16HBE repair ability of human bronchial epithelial cells. Purpose: lentivirus Brg1-sh RNA transfection into human bronchial epithelial cells after 16HBE knockdown Brg1 was used to observe the effect and mechanism of Brg1 on cell proliferation and migration. Methods: to construct lentiviral Brg1 interference vector, expression vector and packaging plasmids with the constructed lentivirus (packaging Mix) were transfected into 293T cells, virus packaging, using the packaged lentivirus (Brg1-sh RNA) and negative control lentivirus infection (Brg1-sc RNA) 16HBE, Blot Q PCR and verified by Western method after stable lines. The cell migration and proliferation ability of two groups were detected by cell scratch test, Transwell compartment and CCK8, and flow cytometry. The two cell lines were detected by fluorescence quantitative PCR and WB, and two other epithelial markers ZO-1 and Occlubin1 were detected by fluorescence quantitative PCR. Dual luciferase reporter gene was used to analyze the interaction between Brg1 and E-cadherin promoter region in two groups. Ch IP-PCR was used to detect the binding strength of 3 promoters in two groups of cells Brg1 and E-cadherin. Results: the cell scratch test and Transwell chamber experiment results showed that Brg1 gene knockdown can promote the migration of airway epithelial cells, CCK8 assay and flow cytometry measuring cell cycle showed that Brg1 gene knockdown can promote airway epithelial cell proliferation ability, proliferation ability is mainly manifested in the S phase of the cell cycle. The results of fluorescence quantitative PCR and WB showed that compared with untransfected 16HBE cell lines and transfected Brg1-sc RNA cell lines, the m RNA level and protein expression level of E-cadherin cells transfected with Brg1-sh RNA increased significantly, while the level of ZO-1 and M did not change significantly. Double luciferase reporter gene showed that compared with cell lines transfected with Brg1-sh RNA, Brg1 had strong binding with -1964/+941bp in promoter region of Brg1-sc RNA, while no significant difference was found in other sequences. Ch IP-PCR results showed that the first segment -86/+60 BP of Brg1 and E-cadherin promoter was the strongest combination in Brg1-sc RNA cell lines, while the other two promoters had no significant difference in two cell lines. Conclusion: the knockdown of Brg1 gene can enhance the migration and proliferation of 16HBE cells, mainly by inhibiting the transcription activation of Brg1 through binding with -86/+60 BP of E-cadherin promoter region.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R562.25

【相似文献】