不同浓度脂多糖对哮喘大鼠气道平滑肌细胞合成分泌功能的影响及其机制的研究
发布时间：2019-06-21 16:04
【摘要】：目的：我们将通过建立哮喘大鼠模型,从细胞水平及整体动物水平研究不同浓度脂多糖(lipopolysaccharide, LPS)刺激哮喘大鼠气道平滑肌细胞(Airway smooth muscle cells, ASMCs)诱导TLR4信号通路激活,对哮喘大鼠ASMCs合成分泌的影响及其机制。 方法：用卵清蛋白(OVA)致敏并激发建立大鼠哮喘模型,分离哮喘大鼠ASMCs进行原代培养,用不同浓度LPS刺激及抗TLR4抗体阻断TLR4。采用酶联免疫吸附测定法(ELISA)检测大鼠ASMCs培养上清液分泌细胞因子IFN-γ、IL-4的情况,逆转录-聚合酶链反应(RT-PCR)检测大鼠ASMCs中TLR4mRNA的表达水平,从而对体外培养的ASMCs在不同浓度LPS刺激后的分泌细胞因子的情况及TLR4表达水平进行研究；本课题还通过建立大鼠哮喘模型,用不同浓度LPS对大鼠进行干预刺激,采用苏木素伊红(HE)染色观察大鼠肺组织病理改变,RT-PCR法检测大鼠气道平滑肌TLR4mRNA的表达水平,探讨不同浓度LPS对哮喘大鼠气道炎症及气道重塑的影响。 1.结果(第一部分实验) (1) ELISA法检测各组哮喘大鼠ASMCs分泌细胞因子：低浓度LPS组(B)IL-4蛋白含量高于空白对照组(A)(P0.01),IFN-Y蛋白含量与空白对照组(A)比较差异无统计学意义(P0.05)；高浓度LPS组(C)IL-4蛋白含量低于空白对照组(A)(P0.01),IFN-γ蛋白含量高于空白对照组(A)(P0.01)；低浓度LPS组(B)IL-4蛋白含量高于高浓度LPS组(C)(P0.01),IFN-γ蛋白含量低于高浓度LPS组(C)(P0.01)；低浓度LPS+TLR4抗体组(D)IL-4蛋白含量低于低浓度LPS组(B)(P0.01),IFN-γ蛋白含量与低浓度LPS组(B)比较差异无统计学意义(P0.05),分别与空白对照组(A)IL-4、IFN-γ含量比较差异无统计学意义(P0.05)；高浓度LPS+TLR4抗体组(E)IL-4蛋白含量高于高浓度LPS组(C)(P0.01),IFN-γ蛋白含量低于高浓度LPS组(B)(P0.01),分别与空白对照组(A)IL-4、IFN-γ含量比较差异无统计学意义(P0.05)。 (2)RT-PCR检测各组哮喘大鼠ASMCs中TLRmRNA表达水平：低浓度LPS组(B)、高浓度LPS组(C)TLR4mRNA表达水平高于空白对照组(A)(P0.01)；高浓度LPS组(C)TLR4mRNA表达水平高于低浓度LPS组(B)(P0.05)；低浓度LPS+TLR4抗体组(D)、高浓度LPS+TLR4抗体组(E)TLR4mRNA表达水平低于空白对照组(A)(P0.01)；低浓度LPS+TLR4抗体组(D)TLR4mRNA表达水平低于低浓度LPS组(B)(P0.01)；高浓度LPS+TLR4抗体组(E)TLR4mRNA表达水平低于高浓度LPS组(C)(P0.01)。 2.结果(第二部分实验) (1)HE染色观察：哮喘组(A组)可见支气管、肺泡及肺间质充斥大量炎症细胞,支气管壁增厚、粘膜层肿胀充血、皱折增多,平滑肌增厚,管腔狭窄。低浓度LPS组(B组)上述炎症改变无明显减轻。高浓度LPS组(C组)上述症状明显减轻。正常对照组(D组)肺组织结构正常,无明显炎症表现。 (2)支气管壁厚度(WAt/Pi)、支气管壁平滑肌厚度(WAm/Pi)、支气管壁平滑肌细胞核数量(N/Pi)：哮喘组(A组)、低浓度LPS组(B组)及高浓度LPS组(C组)支气管壁厚度(WAt/Pi)、支气管壁平滑肌厚度(WAm/Pi)、支气管壁平滑肌细胞核数量(N/Pi)均显著高于正常对照组(D组)(P0.01)；低浓度LPS组(B组)支气管壁厚度(WAt/Pi)、支气管壁平滑肌厚度(WAm/Pi)、支气管壁平滑肌细胞核数量(N/Pi)显著高于哮喘组(A组)(P0.01),高浓度LPS组(C组)支气管壁厚度(WAt/Pi)、支气管壁平滑肌厚度(WAm/Pi)、支气管壁平滑肌细胞核数量(N/Pi)显著低于哮喘组(A组)(P0.01)。 (3)各组大鼠气道阻力比较发现：哮喘组(A组)、低浓度LPS组(B组)、高浓度LPS组(C组)气道阻力均明显高于正常对照组(D组)(P0.01)；高浓度LPS组(C组)气道阻力显著低于哮喘组(A组)(P0.01),低浓度LPS组(B组)与哮喘组(A组)比较差异无统计学意义(P0.05)；高浓度LPS组(C组)气道阻力明显低于低浓度LPS组(B组)(P0.01)。 (4) RT-PCR检测各组哮喘大鼠气道平滑肌中TLRmRNA表达水平：哮喘组(A组)、低浓度LPS组(B组)、高浓度LPS组(C组)明显高于正常对照组(D组)(P0.01)；低浓度LPS组(B组)、高浓度LPS组(C组)气道平滑肌TLR4mRNA表达水平明显高于哮喘组(A组)(P0.01)；高浓度LPS组(C组)气道平滑肌TLR4mRNA表达水平明显高于低浓度LPS组(B组)(P0.05)。 结论： (1)LPS通过激活哮喘大鼠ASMCs表面TLR4,使ASMCs合成分泌Th1/Th2型细胞因子失衡,从而参与哮喘气道炎症改变。 (2)TLR4在哮喘气道炎症及气道重塑中起重要作用,LPS通过激活TLR4在哮喘气道炎症及气道重塑的发病过程中可能发挥双向调控作用。
[Abstract]:Objective: We will study the activation of the TLR4 signaling pathway by establishing an asthma rat model from the level of the cell and the level of the whole animal to study the airway smooth muscle cells (ASMCs) of the asthmatic rats. The effect of ASMCs and its mechanism on the synthesis of ASMCs in asthmatic rats. Methods: The rats were sensitized with ovalbumin (OVA) and excited to establish the model of asthma in rats. ASMCs were isolated from the asthmatic rats. The TLR was blocked by LPS and anti-TLR4 antibodies at different concentrations. 4. The expression of TLR4 mRNA in ASMCs of rats was detected by reverse transcription-polymerase chain reaction (RT-PCR) using an enzyme-linked immunosorbent assay (ELISA) to detect the secretion of cytokines, IFN-1, IL-4 in the rat ASMCs. In this study, the secretion of cytokines and the expression level of TLR4 in cultured ASMCs in vitro were studied. The model of asthma in rats was also established, and the rats were treated with LPS in different concentrations of LPS. The expression level of TLR4 mRNA in the rat airway smooth muscle was detected by hematoxylin eosin (HE) staining, and the expression level of TLR4 mRNA in the airway smooth muscle of the rat was detected by RT-PCR, and the effects of LPS on airway inflammation and airway remodeling in rats with asthma were discussed. in response to that result (first part (1) The levels of IL-4 in low-concentration LPS group were higher than that of blank control group (A) (P0.01), and the content of IFN-Y protein and control group (A) was not statistically significant (P The content of IL-4 in high-concentration LPS group was lower than that of blank control group (A) (P0.01), and the content of IFN-2 protein was higher than that of blank control group (A) (P0.01). The content of IL-4 in low-concentration LPS group was higher than that of high-concentration LPS group (C) (P0.01), and the content of IFN-2 protein was lower than that of high-concentration LPS group (C) (P The content of IL-4 in low-concentration LPS + TLR4 antibody group was lower than that of low-concentration LPS group (B) (P0.01). The content of IL-4 in the high-concentration LPS + TLR4 antibody group was higher than that of the high-concentration LPS group (C) (P0.01). The content of IFN-2 protein was lower than that of the high-concentration LPS group (B) (P0.01). There was no significant difference in the content of IL-4 and IFN-2 in the control group (P (2) The expression level of TLRmRNA in ASMCs was detected by RT-PCR: low-concentration LPS group (B), high-concentration LPS group (C) TLR4 mRNA expression level was higher than that of blank control group (A) (P0.01), and high-concentration LPS group (C) TLR4 mRNA expression level was higher than that of low-concentration LPS group (B) (P0.05); low-concentration LPS + TLR The level of TLR4 mRNA in the high-concentration LPS + TLR4 antibody group was lower than that of the control group (A) (P0.01). The level of TLR4 mRNA in the low-concentration LPS + TLR4 antibody group was lower than that of the low-concentration LPS group (P0.01), and the level of TLR4 mRNA in the high-concentration LPS + TLR4 antibody group was lower than that of the high-concentration LPS group (C). (P0.01). 2. Results (Part 2) (1) HE staining: The bronchial, alveolar and pulmonary interstitial cells in the group of asthma (group A) were filled with a large amount of inflammatory cells, the wall of the bronchi was increased, the swelling of the mucosa was swollen and the number of folds increased. , smooth muscle, narrow lumen, low concentration LPS group (group B) The above-mentioned inflammatory changes were not significantly reduced. The high concentration of LPS groups ( Group C) The above symptoms were significantly reduced. The normal control group (group D) had a lung tissue junction Normal, no obvious inflammation. (2) Bronchial wall thickness (WAt/ Pi), bronchial wall smooth muscle thickness (Wm/ Pi), bronchial wall smooth muscle cell nucleus (N/ Pi): asthma group (group A), low-concentration LPS group (group B) and high-concentration LPS group (group C) The thickness of the tube wall (WAt/ Pi), the thickness of the smooth muscle of the bronchial wall (Wm/ Pi) and the number of the smooth muscle cells of the bronchial wall (N/ Pi) were significantly higher than that in the normal control group (group D) (P0.01); the bronchial wall thickness (Wt/ Pi) and the bronchial wall of the low-concentration LPS group (group B) The smooth muscle thickness (Wm/ Pi) and the number of smooth muscle cells (N/ Pi) in the bronchial wall were significantly higher than that of the asthma group (group A) (P 0.01), the bronchial wall thickness (Wt/ Pi) and the bronchial wall of the high-concentration LPS group (group C). The thickness of smooth muscle (WAm/ Pi) and the number of smooth muscle cells (N/ Pi) in the bronchial wall were significantly lower than that of the control group. The airway resistance in group A (group A), low-concentration LPS group (group B) and high-concentration LPS group (group C) were significantly higher than that in group A. The airway resistance of the high-concentration LPS group (group C) was significantly lower than that of the asthma group (group A) (P0.05). The airway resistance of the high-concentration LPS group (group C) was significantly lower than that of the low-concentration LPS group (group C). The expression of TLRmRNA in the airway smooth muscle of asthmatic rats was detected by RT-PCR (group B) (P 0.01). (4) The expression of TLRmRNA in the airway smooth muscle of each group was detected by RT-PCR: the group of asthma (group A), the low-concentration LPS group (group B) and the high-concentration LPS group (group C) were significantly higher than that in the normal control group (group D) (P The expression of TLR4 mRNA in the airway smooth muscle of the LPS group (group B) and the high-concentration LPS group (group C) was significantly higher than that of the asthma group (group A) (P0.01), and the level of TLR4 mRNA in the high-concentration LPS group (group C) was significantly higher than that of the low-concentration LPS group (group C). Concentration L Conclusion: (1) LPS can secrete Th1/ Th by activating ASMCs surface TLR4 in asthmatic rats (2) TLR4 plays an important role in airway inflammation and airway remodeling in asthma.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R562.25
本文编号：2504197
[Abstract]:Objective: We will study the activation of the TLR4 signaling pathway by establishing an asthma rat model from the level of the cell and the level of the whole animal to study the airway smooth muscle cells (ASMCs) of the asthmatic rats. The effect of ASMCs and its mechanism on the synthesis of ASMCs in asthmatic rats. Methods: The rats were sensitized with ovalbumin (OVA) and excited to establish the model of asthma in rats. ASMCs were isolated from the asthmatic rats. The TLR was blocked by LPS and anti-TLR4 antibodies at different concentrations. 4. The expression of TLR4 mRNA in ASMCs of rats was detected by reverse transcription-polymerase chain reaction (RT-PCR) using an enzyme-linked immunosorbent assay (ELISA) to detect the secretion of cytokines, IFN-1, IL-4 in the rat ASMCs. In this study, the secretion of cytokines and the expression level of TLR4 in cultured ASMCs in vitro were studied. The model of asthma in rats was also established, and the rats were treated with LPS in different concentrations of LPS. The expression level of TLR4 mRNA in the rat airway smooth muscle was detected by hematoxylin eosin (HE) staining, and the expression level of TLR4 mRNA in the airway smooth muscle of the rat was detected by RT-PCR, and the effects of LPS on airway inflammation and airway remodeling in rats with asthma were discussed. in response to that result (first part (1) The levels of IL-4 in low-concentration LPS group were higher than that of blank control group (A) (P0.01), and the content of IFN-Y protein and control group (A) was not statistically significant (P The content of IL-4 in high-concentration LPS group was lower than that of blank control group (A) (P0.01), and the content of IFN-2 protein was higher than that of blank control group (A) (P0.01). The content of IL-4 in low-concentration LPS group was higher than that of high-concentration LPS group (C) (P0.01), and the content of IFN-2 protein was lower than that of high-concentration LPS group (C) (P The content of IL-4 in low-concentration LPS + TLR4 antibody group was lower than that of low-concentration LPS group (B) (P0.01). The content of IL-4 in the high-concentration LPS + TLR4 antibody group was higher than that of the high-concentration LPS group (C) (P0.01). The content of IFN-2 protein was lower than that of the high-concentration LPS group (B) (P0.01). There was no significant difference in the content of IL-4 and IFN-2 in the control group (P (2) The expression level of TLRmRNA in ASMCs was detected by RT-PCR: low-concentration LPS group (B), high-concentration LPS group (C) TLR4 mRNA expression level was higher than that of blank control group (A) (P0.01), and high-concentration LPS group (C) TLR4 mRNA expression level was higher than that of low-concentration LPS group (B) (P0.05); low-concentration LPS + TLR The level of TLR4 mRNA in the high-concentration LPS + TLR4 antibody group was lower than that of the control group (A) (P0.01). The level of TLR4 mRNA in the low-concentration LPS + TLR4 antibody group was lower than that of the low-concentration LPS group (P0.01), and the level of TLR4 mRNA in the high-concentration LPS + TLR4 antibody group was lower than that of the high-concentration LPS group (C). (P0.01). 2. Results (Part 2) (1) HE staining: The bronchial, alveolar and pulmonary interstitial cells in the group of asthma (group A) were filled with a large amount of inflammatory cells, the wall of the bronchi was increased, the swelling of the mucosa was swollen and the number of folds increased. , smooth muscle, narrow lumen, low concentration LPS group (group B) The above-mentioned inflammatory changes were not significantly reduced. The high concentration of LPS groups ( Group C) The above symptoms were significantly reduced. The normal control group (group D) had a lung tissue junction Normal, no obvious inflammation. (2) Bronchial wall thickness (WAt/ Pi), bronchial wall smooth muscle thickness (Wm/ Pi), bronchial wall smooth muscle cell nucleus (N/ Pi): asthma group (group A), low-concentration LPS group (group B) and high-concentration LPS group (group C) The thickness of the tube wall (WAt/ Pi), the thickness of the smooth muscle of the bronchial wall (Wm/ Pi) and the number of the smooth muscle cells of the bronchial wall (N/ Pi) were significantly higher than that in the normal control group (group D) (P0.01); the bronchial wall thickness (Wt/ Pi) and the bronchial wall of the low-concentration LPS group (group B) The smooth muscle thickness (Wm/ Pi) and the number of smooth muscle cells (N/ Pi) in the bronchial wall were significantly higher than that of the asthma group (group A) (P 0.01), the bronchial wall thickness (Wt/ Pi) and the bronchial wall of the high-concentration LPS group (group C). The thickness of smooth muscle (WAm/ Pi) and the number of smooth muscle cells (N/ Pi) in the bronchial wall were significantly lower than that of the control group. The airway resistance in group A (group A), low-concentration LPS group (group B) and high-concentration LPS group (group C) were significantly higher than that in group A. The airway resistance of the high-concentration LPS group (group C) was significantly lower than that of the asthma group (group A) (P0.05). The airway resistance of the high-concentration LPS group (group C) was significantly lower than that of the low-concentration LPS group (group C). The expression of TLRmRNA in the airway smooth muscle of asthmatic rats was detected by RT-PCR (group B) (P 0.01). (4) The expression of TLRmRNA in the airway smooth muscle of each group was detected by RT-PCR: the group of asthma (group A), the low-concentration LPS group (group B) and the high-concentration LPS group (group C) were significantly higher than that in the normal control group (group D) (P The expression of TLR4 mRNA in the airway smooth muscle of the LPS group (group B) and the high-concentration LPS group (group C) was significantly higher than that of the asthma group (group A) (P0.01), and the level of TLR4 mRNA in the high-concentration LPS group (group C) was significantly higher than that of the low-concentration LPS group (group C). Concentration L Conclusion: (1) LPS can secrete Th1/ Th by activating ASMCs surface TLR4 in asthmatic rats (2) TLR4 plays an important role in airway inflammation and airway remodeling in asthma.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R562.25
【参考文献】
相关期刊论文 前3条
1 韦江红;莫碧文;黄剑伟;徐青;林武州;;TOLL样受体4对哮喘气道平滑肌细胞分泌IL-5、IL-8的影响[J];山东医药;2011年03期
2 郭菲,汪泱,王共先,李国辉,邢娟娟,彭燕,黄学明;内毒素/脂多糖对小鼠腹腔巨噬细胞膜Toll样受体2及4表达的影响[J];中华烧伤杂志;2005年05期
3 ;Upregulated functional expression of Toll like receptor 4 in mesenchymal stem cells induced by lipopolysaccharide[J];Chinese Medical Journal;2007年19期
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