YKL-40誘導人支氣管上皮細胞發(fā)生EMT效應及誘導HMGB-1表達的研究
發(fā)布時間：2019-07-05 14:26
【摘要】：背景和目的:YKL-40是一種進化上高度保守的缺乏甲殼素酶活性的甲殼素酶蛋白家族成員之一,是新近發(fā)現(xiàn)的炎癥因子。近年來,多項研究證實YKL-40蛋白在炎癥、組織重塑的病理狀態(tài)中發(fā)揮著重要的作用。支氣管哮喘是一種慢性氣道炎癥性疾病,以氣道重塑為主要病理學特征之一。目前證實YKL-40與支氣管哮喘發(fā)病過程中的氣道重構有密切關系。上皮-間質轉化是氣道重構的發(fā)生機制之一。有報道指出上皮-間質轉化的這一病理狀態(tài)能夠促進肺纖維化和哮喘氣道重構的發(fā)生。那么,YKL-40能否誘導支氣管上皮細胞發(fā)生間質樣細胞改變,從而進一步促進氣道重構發(fā)生、發(fā)展,有待進一步的研究、探索。HMGB-1是一種非組蛋白,屬于“損傷相關模式分子”(DAMP)家族的一員,細胞處于應激狀態(tài)時可觸發(fā)HMGB-1的主動或被動釋放。近年來,HMGB-1與支氣管哮喘發(fā)病的相關性得到越來越多的研究證實。支氣管哮喘的發(fā)病具體機制尚不得而知。由于YKL-40和HMGB-1這兩種物質都是哮喘發(fā)病過程中的重要調節(jié)因子,它們之間可能存在某種聯(lián)系,從而共同參與哮喘的發(fā)病過程。因此,本課題的主要研究目的是了解YKL-40蛋白在體外對支氣管上皮細胞(Beas-2B細胞)的影響,探討其能否誘導Beas-2B細胞發(fā)生上皮間質轉化(EMT效應),同時明確其能否促進Beas-2B細胞表達和分泌HMGB-1。方法:我們體外成功復蘇并培養(yǎng)了人支氣管上皮細胞(Beas-2B細胞),以用來探討YKL-40誘導支氣管上皮細胞發(fā)生EMT效應和HMGB-1的作用關系;用不同濃度的YKL-40蛋白刺激Beas-2B細胞24h后,在倒置顯微鏡下觀察Beas-2B細胞的形態(tài)學變化;同時采用細胞免疫熒光實驗檢測并比較細胞中上皮標志物E-cadherin和間質相關的標志物N-cadherin、Vimentin、α-SMA的熒光強弱變化;后進一步采用Western-Blot法檢測E-cadherin、N-cadherin、Vimentin和Desmin的蛋白表達水平;另外,Beas-2B細胞接受不同濃度的YKL-40蛋白刺激12h后,采用Real-time RT-PCR法測定細胞提取蛋白中的E-cadherin、N-cadherin、Vimentin和Desmin的m RNA表達水平。后為進一步研究YKL-40對HMGB-1的影響,我們分別采用Elisa、real-time RT-PCR和Western-Blot法檢測不同濃度的YKL-40作用于Beas-2B后HMGB-1的表達情況。按照實驗設計要求配制多種不同濃度的YKL-40蛋白溶液,并充分混勻,分別于六孔板每孔中加入一定體積濃度為1μg/mL的YKL-40蛋白使得各孔的終濃度分別為0ng/mL、10 ng/mL、100 ng/mL和400 ng/mL,混勻后放入細胞培養(yǎng)箱中刺激培養(yǎng)24h。收集各孔培養(yǎng)液上清、細胞裂解液,并分別予以詳細注明、命名后待用。隨后采用Elisa方法檢測上清HMGB-1水平;采用Western Blot法檢測HMGB-1、E-cadherin、N-cadherin、Vimentin和Desmin的蛋白表達水平;同時采用熒光實時定量PCR法檢測HMGB-1、E-cadherin、N-cadherin、Vimentin和Desmin的mRNA表達水平;細胞免疫熒光染色法觀察細胞內E-cadherin、N-cadherin、Vimentin和α-SMA的熒光強弱變化。同時在倒置顯微鏡下可以觀察各組不同濃度的YKL-40作用下支氣管上皮細胞(Beas-2B)的形態(tài)學變化。結果:我們在顯微鏡下觀察到,Bease-2B細胞受到不同濃度的YKL-40蛋白刺激后,其上皮細胞形態(tài)向間質細胞樣形態(tài)轉化,多數(shù)細胞由原來的上皮細胞“鋪路石樣”形態(tài)轉化為梭形、紡錘體形的間質細胞改變;同時,細胞免疫熒光染色結果表明,YKL-40能夠上調間質標志物N-cadherin、α-SMA、Vimentin的表達,而上皮標志物E-cadherin的表達則呈降低趨勢;隨后的RT-PCR和Western-Blot實驗也進一步證實了上述的結果,以上結果說明YKL-40蛋白可以濃度依賴性方式促進Beas-2B細胞發(fā)生EMT效應。此外,我們采用Elisa、RT-PCR和Western-Blot實驗說明,YKL-40能夠呈濃度依賴性的方式促進HMGB-1的表達。結論:YKL-40能夠誘導支氣管上皮細胞發(fā)生EMT效應,同時能夠促進支氣管上皮細胞表達和分泌HMGB-1。該項研究提示YKL-40與HMGB-1存在聯(lián)系,有望為支氣管哮喘發(fā)病機制的研究提供新的證據(jù)。
文內圖片：
圖片說明：低倍鏡（10×40倍）觀察Beas-2B細胞受YKL-40蛋白刺激后形態(tài)發(fā)生改變
[Abstract]:BACKGROUND & OBJECTIVE: YKL-40 is one of the members of the family of the family members of the family of chitin, which is highly conserved in evolution, and is a newly discovered inflammatory factor. In recent years, several studies have shown that the YKL-40 protein plays an important role in the pathological state of inflammation and tissue remodeling. Bronchial asthma is a chronic airway inflammatory disease, which is one of the main pathological features of airway remodeling. It is confirmed that YKL-40 is closely related to airway remodeling in the pathogenesis of bronchial asthma. Epithelial-mesenchymal transition is one of the mechanisms of airway remodeling. It is reported that this pathological state of epithelial-mesenchymal transition can promote the occurrence of pulmonary fibrosis and airway remodeling in asthma. Therefore, the ability of YKL-40 to induce the changes of the interstitial-like cells in the bronchial epithelial cells, thus further promoting the development of the airway remodeling, is to be further studied and explored. HMGB-1 is a non-histone, which is part of the "damage-related model molecule" (DAMP) family and can trigger the active or passive release of the HMGB-1 when the cell is in a normal state. In recent years, the correlation between HMGB-1 and bronchial asthma is more and more confirmed. The mechanism of the pathogenesis of bronchial asthma is unknown. Since both YKL-40 and HMGB-1 are important regulators in the pathogenesis of asthma, there may be some association between them, thus participating in the pathogenesis of asthma. Therefore, the main purpose of this study is to understand the effect of YKL-40 protein on the bronchial epithelial cells (Beas-2B cells) in vitro, to explore whether it can induce the epithelial-mesenchymal transition (EMT effect) of the Beas-2B cells, and to determine whether it can promote the expression of the Beas-2B cells and to secrete the HMGB-1. Methods: We successfully recovered and cultured human bronchial epithelial cells (Beas-2B cells) in vitro to investigate the effect of YKL-40 on the development of EMT and HMGB-1 in the bronchial epithelial cells. After 24 h of Beas-2B cells stimulated with YKL-40 protein at different concentrations, The morphological changes of the cells of the Beas-2B cells were observed under an inverted microscope, and the changes of the fluorescence intensity of E-cadherin, Vimentin, and E-SMA in the cells were detected and compared with the cell immunofluorescence assay, and the E-cadherin, N-cadherin was further detected by the Western-Blot method. In addition, the expression levels of E-cadherin, N-cadherin, Vimentin and Desmin in the cell-extracted protein were determined by the Real-time RT-PCR method after 12 h of YKL-40 protein stimulation with different concentrations of the Beas-2B cells. In order to further study the effect of YKL-40 on HMGB-1, the expression of HGB-1 after Beas-2B was detected by Elisa, rel-time RT-PCR and Western-Blot method, respectively. The YKL-40 protein solution with a plurality of different concentrations is prepared according to the experimental design requirements, and the YKL-40 protein with a volume concentration of 1. mu. g/ mL is added into each well of the six-well plate, so that the final concentration of each well is 0 ng/ mL,10 ng/ mL,100 ng/ mL and 400 ng/ mL, respectively. And the mixture is put into a cell culture box for stimulation and culture for 24 hours. The supernatant of each well culture solution and the cell lysis solution shall be collected and specified in detail and be used for later use. The expression level of HMGB-1, E-cadherin, N-cadherin, Vimentin and Desmin was detected by Western Blot method, and the mRNA expression levels of HMGB-1, E-cadherin, N-cadherin, Vimentin and Desmin were detected by fluorescent real-time quantitative PCR. E-cadherin, N-cadherin, The changes of the fluorescence intensity of Vimentin and SMA-SMA. At the same time, the morphological changes of the bronchial epithelial cells (Beas-2B) under different concentrations of YKL-40 were observed under the inverted microscope. Results: We observed under the microscope that the cells of the Bease-2B cells were stimulated by YKL-40 protein at different concentrations. The morphology of the epithelial cells of the cells was transformed into the mesenchymal cells. Most of the cells were transformed into the fusiform and spindle-shaped interstitial cells from the original epithelial cell "paving stone sample". At the same time, The results showed that YKL-40 could upregulate the expression of N-cadherin, VEGF-SMA and Vimentin, and the expression of E-cadherin in E-cadherin was decreased. The results were also confirmed by RT-PCR and Western-Blot. The above results show that the YKL-40 protein can promote the EMT effect of the Beas-2B cells in a concentration-dependent manner. In addition, we used Elisa, RT-PCR and Western-Blot to show that YKL-40 can promote the expression of HMGB-1 in a concentration-dependent manner. Conclusion: YKL-40 can induce the EMT effect of bronchial epithelial cells, and can promote the expression and secretion of HMGB-1 in the bronchial epithelial cells. The study suggests that YKL-40 is associated with HMGB-1 and is expected to provide new evidence for the study of the pathogenesis of bronchial asthma.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R562.25
本文編號：2510588
文內圖片：
圖片說明：低倍鏡（10×40倍）觀察Beas-2B細胞受YKL-40蛋白刺激后形態(tài)發(fā)生改變
[Abstract]:BACKGROUND & OBJECTIVE: YKL-40 is one of the members of the family of the family members of the family of chitin, which is highly conserved in evolution, and is a newly discovered inflammatory factor. In recent years, several studies have shown that the YKL-40 protein plays an important role in the pathological state of inflammation and tissue remodeling. Bronchial asthma is a chronic airway inflammatory disease, which is one of the main pathological features of airway remodeling. It is confirmed that YKL-40 is closely related to airway remodeling in the pathogenesis of bronchial asthma. Epithelial-mesenchymal transition is one of the mechanisms of airway remodeling. It is reported that this pathological state of epithelial-mesenchymal transition can promote the occurrence of pulmonary fibrosis and airway remodeling in asthma. Therefore, the ability of YKL-40 to induce the changes of the interstitial-like cells in the bronchial epithelial cells, thus further promoting the development of the airway remodeling, is to be further studied and explored. HMGB-1 is a non-histone, which is part of the "damage-related model molecule" (DAMP) family and can trigger the active or passive release of the HMGB-1 when the cell is in a normal state. In recent years, the correlation between HMGB-1 and bronchial asthma is more and more confirmed. The mechanism of the pathogenesis of bronchial asthma is unknown. Since both YKL-40 and HMGB-1 are important regulators in the pathogenesis of asthma, there may be some association between them, thus participating in the pathogenesis of asthma. Therefore, the main purpose of this study is to understand the effect of YKL-40 protein on the bronchial epithelial cells (Beas-2B cells) in vitro, to explore whether it can induce the epithelial-mesenchymal transition (EMT effect) of the Beas-2B cells, and to determine whether it can promote the expression of the Beas-2B cells and to secrete the HMGB-1. Methods: We successfully recovered and cultured human bronchial epithelial cells (Beas-2B cells) in vitro to investigate the effect of YKL-40 on the development of EMT and HMGB-1 in the bronchial epithelial cells. After 24 h of Beas-2B cells stimulated with YKL-40 protein at different concentrations, The morphological changes of the cells of the Beas-2B cells were observed under an inverted microscope, and the changes of the fluorescence intensity of E-cadherin, Vimentin, and E-SMA in the cells were detected and compared with the cell immunofluorescence assay, and the E-cadherin, N-cadherin was further detected by the Western-Blot method. In addition, the expression levels of E-cadherin, N-cadherin, Vimentin and Desmin in the cell-extracted protein were determined by the Real-time RT-PCR method after 12 h of YKL-40 protein stimulation with different concentrations of the Beas-2B cells. In order to further study the effect of YKL-40 on HMGB-1, the expression of HGB-1 after Beas-2B was detected by Elisa, rel-time RT-PCR and Western-Blot method, respectively. The YKL-40 protein solution with a plurality of different concentrations is prepared according to the experimental design requirements, and the YKL-40 protein with a volume concentration of 1. mu. g/ mL is added into each well of the six-well plate, so that the final concentration of each well is 0 ng/ mL,10 ng/ mL,100 ng/ mL and 400 ng/ mL, respectively. And the mixture is put into a cell culture box for stimulation and culture for 24 hours. The supernatant of each well culture solution and the cell lysis solution shall be collected and specified in detail and be used for later use. The expression level of HMGB-1, E-cadherin, N-cadherin, Vimentin and Desmin was detected by Western Blot method, and the mRNA expression levels of HMGB-1, E-cadherin, N-cadherin, Vimentin and Desmin were detected by fluorescent real-time quantitative PCR. E-cadherin, N-cadherin, The changes of the fluorescence intensity of Vimentin and SMA-SMA. At the same time, the morphological changes of the bronchial epithelial cells (Beas-2B) under different concentrations of YKL-40 were observed under the inverted microscope. Results: We observed under the microscope that the cells of the Bease-2B cells were stimulated by YKL-40 protein at different concentrations. The morphology of the epithelial cells of the cells was transformed into the mesenchymal cells. Most of the cells were transformed into the fusiform and spindle-shaped interstitial cells from the original epithelial cell "paving stone sample". At the same time, The results showed that YKL-40 could upregulate the expression of N-cadherin, VEGF-SMA and Vimentin, and the expression of E-cadherin in E-cadherin was decreased. The results were also confirmed by RT-PCR and Western-Blot. The above results show that the YKL-40 protein can promote the EMT effect of the Beas-2B cells in a concentration-dependent manner. In addition, we used Elisa, RT-PCR and Western-Blot to show that YKL-40 can promote the expression of HMGB-1 in a concentration-dependent manner. Conclusion: YKL-40 can induce the EMT effect of bronchial epithelial cells, and can promote the expression and secretion of HMGB-1 in the bronchial epithelial cells. The study suggests that YKL-40 is associated with HMGB-1 and is expected to provide new evidence for the study of the pathogenesis of bronchial asthma.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R562.25
【參考文獻】
相關期刊論文 前1條
1 ;Inhibition of high-mobility group box 1 expression by siRNA in rat hepatic stellate cells[J];World Journal of Gastroenterology;2011年36期
，本文編號：2510588
本文鏈接：http://sikaile.net/yixuelunwen/nfm/2510588.html
最近更新
教材專著
