本文關(guān)鍵詞：染色質(zhì)重塑因子Brg1通過(guò)E-cadherin影響哮喘氣道上皮完整性的研究　出處：《重慶醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 哮喘 Brg1 氣道上皮 哮喘 Brg1 E-cadherin AHR 氣道炎癥 16HBE Brg1 E-cadherin ZO-1 氣道上皮修復(fù)

【摘要】：第一部分 Brg1在哮喘C57BL/6小鼠的肺組織和氣道上皮異常表達(dá)目的:觀察染色質(zhì)重塑因子Brg1基因在哮喘C57BL/6小鼠的肺組織和氣道上皮的異常表達(dá),了解Brg1和哮喘的相關(guān)性。方法:將小鼠分為兩組:正常對(duì)照組(control)和哮喘組(asthma)。小鼠在最后一次霧化48 h內(nèi)依次進(jìn)行AHR檢測(cè)、支氣管肺泡灌洗和BALF細(xì)胞計(jì)數(shù),同時(shí)取BALF細(xì)胞沉渣涂片用于細(xì)胞分類計(jì)數(shù),取肺病理組織用于HE染色、E-cadherin免疫組化染色,取肺組織標(biāo)本用于提m RNA和蛋白進(jìn)行Q-PCR和WB檢測(cè)。結(jié)果:肺組織中Brg1的m RNA和蛋白水平在C57BL/6小鼠哮喘組明顯升高,抗Brg1免疫組化染色顯示Brg1在哮喘組的氣道上皮上表達(dá)增多。結(jié)論:哮喘中Brg1的m RNA和蛋白水平均有明顯升高,提示Brg1和哮喘有一定的相關(guān)性。同時(shí)發(fā)現(xiàn)Brg1在哮喘組的氣道上皮上高表達(dá),提示Brg1與氣道上皮可能有一定的關(guān)系,進(jìn)一步對(duì)Brg1進(jìn)行研究也許可以發(fā)現(xiàn)哮喘氣道上皮損傷后修復(fù)的其他機(jī)制。第二 部分Brg1基因敲除對(duì)C57BL/6哮喘小鼠氣道上皮完整性的影響目的:觀察Brg1基因?qū)ο∈驛HR、氣道炎癥和上皮marker E-cadherin的影響。方法:將Cre重組酶轉(zhuǎn)基因小鼠(SFTPC-rt TA/(Tet O)7)與lox P轉(zhuǎn)基因雜合子小鼠(Brg1lox P/lox P)雜交后篩出純合型Brg1敲除鼠(Brg1~(-/-))。將哮喘小鼠分為四組:野生型小鼠正常對(duì)照組(WT ctrl),野生型小鼠哮喘組(WT asthma),Brg1基因敲除鼠正常對(duì)照組(Brg1~(-/-)ctrl),Brg1基因敲除鼠哮喘組(Brg1~(-/-)asthma)。小鼠在最后一次霧化48 h內(nèi)依次進(jìn)行有創(chuàng)肺功能氣道高反應(yīng)性AHR檢測(cè)、支氣管肺泡灌洗和BALF細(xì)胞計(jì)數(shù),同時(shí),BALF細(xì)胞沉渣涂片用于細(xì)胞分類計(jì)數(shù),肺病理組織用于HE染色、E-cadherin免疫組化染色,取肺組織標(biāo)本用于提m RNA和蛋白進(jìn)行熒光定量PCR和WB檢測(cè)。結(jié)果:有創(chuàng)肺功能AHR檢測(cè)、BALF細(xì)胞計(jì)數(shù)結(jié)果顯示,與WT asthma組相比,Brg1~(-/-)asthma組的AHR明顯下降,BALF中氣道炎癥細(xì)胞和嗜酸性細(xì)胞的數(shù)量均有明顯降低。HE染色明確顯示Brg1~(-/-)asthma組氣道周圍炎癥細(xì)胞的浸潤(rùn)明顯減輕,氣道上皮的完整性得到一定程度的改善�？股掀arker E-cadherin免疫組化染色顯示與WT asthma組的E-cadherin低表達(dá)相比,E-cadherin在Brg1~(-/-)asthma組的氣道上皮中表達(dá)增高,而B(niǎo)rg1~(-/-)Ctrl組的E-cadherin的表達(dá)與WT Ctrl組相比無(wú)明顯差異。氣道炎癥細(xì)胞、嗜酸性細(xì)胞數(shù)量與AHR指標(biāo)LR均呈相關(guān)性。結(jié)論:Brg1基因的敲除可以明顯降低C57BL/6哮喘小鼠的AHR、BALF中氣道炎癥細(xì)胞和嗜酸性粒細(xì)胞的數(shù)量和氣道周圍炎癥細(xì)胞的浸潤(rùn),同時(shí)增強(qiáng)了氣道上皮marker E-cadherin的表達(dá),提示Brg1基因的敲除可以增強(qiáng)哮喘氣道上皮的完整性。第三部分 Brg1基因敲低對(duì)人支氣管上皮細(xì)胞16HBE的修復(fù)能力的機(jī)制研究目的:利用慢病毒Brg1-sh RNA轉(zhuǎn)染人支氣管上皮細(xì)胞16HBE敲低Brg1后,觀察Brg1對(duì)細(xì)胞增殖和遷移能力的影響及機(jī)制研究。方法:構(gòu)建慢病毒Brg1干擾載體后,用構(gòu)建的慢病毒表達(dá)載體和包裝質(zhì)粒(packaging mix)共轉(zhuǎn)染293T細(xì)胞,包裝病毒,用包裝好的慢病毒(Brg1-sh RNA)以及慢病毒陰性對(duì)照(Brg1-sc RNA)感染16HBE,通過(guò)Western Blot和q PCR方法驗(yàn)證后得到穩(wěn)定株。分別用細(xì)胞劃痕實(shí)驗(yàn)、Transwell小室和CCK8、流式測(cè)細(xì)胞周期技術(shù)檢測(cè)兩組細(xì)胞株的遷移和增殖能力。將兩種細(xì)胞株進(jìn)行E-cadherin的熒光定量PCR和WB檢測(cè),同時(shí)熒光定量PCR檢測(cè)另外兩個(gè)上皮markers ZO-1和Occlubin1。雙重?zé)晒馑孛笀?bào)告基因分析兩組細(xì)胞Brg1與E-cadherin的啟動(dòng)子區(qū)有無(wú)相互作用,Ch IP-PCR檢測(cè)兩組細(xì)胞Brg1與E-cadherin的3個(gè)啟動(dòng)子區(qū)序列的結(jié)合強(qiáng)度。結(jié)果:細(xì)胞劃痕實(shí)驗(yàn)和Transwell小室實(shí)驗(yàn)結(jié)果顯示Brg1基因的敲低可以促進(jìn)氣道上皮細(xì)胞的遷移能力,CCK8檢測(cè)和流式測(cè)細(xì)胞周期結(jié)果顯示Brg1基因的敲低可以促進(jìn)氣道上皮細(xì)胞的增殖能力,增殖能力增強(qiáng)主要表現(xiàn)為細(xì)胞周期的S期增高。熒光定量PCR和WB的結(jié)果顯示,與未轉(zhuǎn)染的16HBE細(xì)胞株和轉(zhuǎn)染Brg1-sc RNA的細(xì)胞株相比較,轉(zhuǎn)染Brg1-sh RNA的細(xì)胞株E-cadherin的m RNA水平和蛋白表達(dá)水平明顯增高,而ZO-1和Occlubin1的m RNA水平無(wú)明顯改變。雙重?zé)晒馑孛笀?bào)告基因顯示,與轉(zhuǎn)染Brg1-sh RNA的細(xì)胞株相比,轉(zhuǎn)染Brg1-sc RNA的細(xì)胞株中Brg1與E-cadherin的啟動(dòng)子區(qū)的-1964/+941bp有較強(qiáng)結(jié)合,而其它序列無(wú)明顯差異。Ch IP-PCR結(jié)果顯示Brg1與E-cadherin的啟動(dòng)子區(qū)的第1段序列-86/+60 bp在轉(zhuǎn)染Brg1-sc RNA的細(xì)胞株中結(jié)合最強(qiáng),而另外兩個(gè)啟動(dòng)子區(qū)序列在兩組細(xì)胞株中無(wú)明顯差異。結(jié)論:Brg1基因敲低后可以提高16HBE細(xì)胞的遷移和增殖能力,主要是Brg1基因通過(guò)與E-cadherin啟動(dòng)子區(qū)的-86/+60 bp結(jié)合來(lái)抑制E-cadherin的轉(zhuǎn)錄激活。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R562.25

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