YKL-40诱导人支气管上皮细胞发生EMT效应及诱导HMGB-1表达的研究
发布时间：2019-07-05 14:26
【摘要】：背景和目的:YKL-40是一种进化上高度保守的缺乏甲壳素酶活性的甲壳素酶蛋白家族成员之一,是新近发现的炎症因子。近年来,多项研究证实YKL-40蛋白在炎症、组织重塑的病理状态中发挥着重要的作用。支气管哮喘是一种慢性气道炎症性疾病,以气道重塑为主要病理学特征之一。目前证实YKL-40与支气管哮喘发病过程中的气道重构有密切关系。上皮-间质转化是气道重构的发生机制之一。有报道指出上皮-间质转化的这一病理状态能够促进肺纤维化和哮喘气道重构的发生。那么,YKL-40能否诱导支气管上皮细胞发生间质样细胞改变,从而进一步促进气道重构发生、发展,有待进一步的研究、探索。HMGB-1是一种非组蛋白,属于“损伤相关模式分子”(DAMP)家族的一员,细胞处于应激状态时可触发HMGB-1的主动或被动释放。近年来,HMGB-1与支气管哮喘发病的相关性得到越来越多的研究证实。支气管哮喘的发病具体机制尚不得而知。由于YKL-40和HMGB-1这两种物质都是哮喘发病过程中的重要调节因子,它们之间可能存在某种联系,从而共同参与哮喘的发病过程。因此,本课题的主要研究目的是了解YKL-40蛋白在体外对支气管上皮细胞(Beas-2B细胞)的影响,探讨其能否诱导Beas-2B细胞发生上皮间质转化(EMT效应),同时明确其能否促进Beas-2B细胞表达和分泌HMGB-1。方法:我们体外成功复苏并培养了人支气管上皮细胞(Beas-2B细胞),以用来探讨YKL-40诱导支气管上皮细胞发生EMT效应和HMGB-1的作用关系;用不同浓度的YKL-40蛋白刺激Beas-2B细胞24h后,在倒置显微镜下观察Beas-2B细胞的形态学变化;同时采用细胞免疫荧光实验检测并比较细胞中上皮标志物E-cadherin和间质相关的标志物N-cadherin、Vimentin、α-SMA的荧光强弱变化;后进一步采用Western-Blot法检测E-cadherin、N-cadherin、Vimentin和Desmin的蛋白表达水平;另外,Beas-2B细胞接受不同浓度的YKL-40蛋白刺激12h后,采用Real-time RT-PCR法测定细胞提取蛋白中的E-cadherin、N-cadherin、Vimentin和Desmin的m RNA表达水平。后为进一步研究YKL-40对HMGB-1的影响,我们分别采用Elisa、real-time RT-PCR和Western-Blot法检测不同浓度的YKL-40作用于Beas-2B后HMGB-1的表达情况。按照实验设计要求配制多种不同浓度的YKL-40蛋白溶液,并充分混匀,分别于六孔板每孔中加入一定体积浓度为1μg/mL的YKL-40蛋白使得各孔的终浓度分别为0ng/mL、10 ng/mL、100 ng/mL和400 ng/mL,混匀后放入细胞培养箱中刺激培养24h。收集各孔培养液上清、细胞裂解液,并分别予以详细注明、命名后待用。随后采用Elisa方法检测上清HMGB-1水平;采用Western Blot法检测HMGB-1、E-cadherin、N-cadherin、Vimentin和Desmin的蛋白表达水平;同时采用荧光实时定量PCR法检测HMGB-1、E-cadherin、N-cadherin、Vimentin和Desmin的mRNA表达水平;细胞免疫荧光染色法观察细胞内E-cadherin、N-cadherin、Vimentin和α-SMA的荧光强弱变化。同时在倒置显微镜下可以观察各组不同浓度的YKL-40作用下支气管上皮细胞(Beas-2B)的形态学变化。结果:我们在显微镜下观察到,Bease-2B细胞受到不同浓度的YKL-40蛋白刺激后,其上皮细胞形态向间质细胞样形态转化,多数细胞由原来的上皮细胞“铺路石样”形态转化为梭形、纺锤体形的间质细胞改变;同时,细胞免疫荧光染色结果表明,YKL-40能够上调间质标志物N-cadherin、α-SMA、Vimentin的表达,而上皮标志物E-cadherin的表达则呈降低趋势;随后的RT-PCR和Western-Blot实验也进一步证实了上述的结果,以上结果说明YKL-40蛋白可以浓度依赖性方式促进Beas-2B细胞发生EMT效应。此外,我们采用Elisa、RT-PCR和Western-Blot实验说明,YKL-40能够呈浓度依赖性的方式促进HMGB-1的表达。结论:YKL-40能够诱导支气管上皮细胞发生EMT效应,同时能够促进支气管上皮细胞表达和分泌HMGB-1。该项研究提示YKL-40与HMGB-1存在联系,有望为支气管哮喘发病机制的研究提供新的证据。
文内图片：
图片说明：低倍镜（10×40倍）观察Beas-2B细胞受YKL-40蛋白刺激后形态发生改变
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25
本文编号：2510588
文内图片：
图片说明：低倍镜（10×40倍）观察Beas-2B细胞受YKL-40蛋白刺激后形态发生改变
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25
【参考文献】
相关期刊论文 前1条
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
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