不同濃度脂多糖對哮喘大鼠氣道平滑肌細胞合成分泌功能的影響及其機制的研究
發(fā)布時間：2019-06-21 16:04
【摘要】：目的：我們將通過建立哮喘大鼠模型,從細胞水平及整體動物水平研究不同濃度脂多糖(lipopolysaccharide, LPS)刺激哮喘大鼠氣道平滑肌細胞(Airway smooth muscle cells, ASMCs)誘導(dǎo)TLR4信號通路激活,對哮喘大鼠ASMCs合成分泌的影響及其機制。 方法：用卵清蛋白(OVA)致敏并激發(fā)建立大鼠哮喘模型,分離哮喘大鼠ASMCs進行原代培養(yǎng),用不同濃度LPS刺激及抗TLR4抗體阻斷TLR4。采用酶聯(lián)免疫吸附測定法(ELISA)檢測大鼠ASMCs培養(yǎng)上清液分泌細胞因子IFN-γ、IL-4的情況,逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)檢測大鼠ASMCs中TLR4mRNA的表達水平,從而對體外培養(yǎng)的ASMCs在不同濃度LPS刺激后的分泌細胞因子的情況及TLR4表達水平進行研究；本課題還通過建立大鼠哮喘模型,用不同濃度LPS對大鼠進行干預(yù)刺激,采用蘇木素伊紅(HE)染色觀察大鼠肺組織病理改變,RT-PCR法檢測大鼠氣道平滑肌TLR4mRNA的表達水平,探討不同濃度LPS對哮喘大鼠氣道炎癥及氣道重塑的影響。 1.結(jié)果(第一部分實驗) (1) ELISA法檢測各組哮喘大鼠ASMCs分泌細胞因子：低濃度LPS組(B)IL-4蛋白含量高于空白對照組(A)(P0.01),IFN-Y蛋白含量與空白對照組(A)比較差異無統(tǒng)計學意義(P0.05)；高濃度LPS組(C)IL-4蛋白含量低于空白對照組(A)(P0.01),IFN-γ蛋白含量高于空白對照組(A)(P0.01)；低濃度LPS組(B)IL-4蛋白含量高于高濃度LPS組(C)(P0.01),IFN-γ蛋白含量低于高濃度LPS組(C)(P0.01)；低濃度LPS+TLR4抗體組(D)IL-4蛋白含量低于低濃度LPS組(B)(P0.01),IFN-γ蛋白含量與低濃度LPS組(B)比較差異無統(tǒng)計學意義(P0.05),分別與空白對照組(A)IL-4、IFN-γ含量比較差異無統(tǒng)計學意義(P0.05)；高濃度LPS+TLR4抗體組(E)IL-4蛋白含量高于高濃度LPS組(C)(P0.01),IFN-γ蛋白含量低于高濃度LPS組(B)(P0.01),分別與空白對照組(A)IL-4、IFN-γ含量比較差異無統(tǒng)計學意義(P0.05)。 (2)RT-PCR檢測各組哮喘大鼠ASMCs中TLRmRNA表達水平：低濃度LPS組(B)、高濃度LPS組(C)TLR4mRNA表達水平高于空白對照組(A)(P0.01)；高濃度LPS組(C)TLR4mRNA表達水平高于低濃度LPS組(B)(P0.05)；低濃度LPS+TLR4抗體組(D)、高濃度LPS+TLR4抗體組(E)TLR4mRNA表達水平低于空白對照組(A)(P0.01)；低濃度LPS+TLR4抗體組(D)TLR4mRNA表達水平低于低濃度LPS組(B)(P0.01)；高濃度LPS+TLR4抗體組(E)TLR4mRNA表達水平低于高濃度LPS組(C)(P0.01)。 2.結(jié)果(第二部分實驗) (1)HE染色觀察：哮喘組(A組)可見支氣管、肺泡及肺間質(zhì)充斥大量炎癥細胞,支氣管壁增厚、粘膜層腫脹充血、皺折增多,平滑肌增厚,管腔狹窄。低濃度LPS組(B組)上述炎癥改變無明顯減輕。高濃度LPS組(C組)上述癥狀明顯減輕。正常對照組(D組)肺組織結(jié)構(gòu)正常,無明顯炎癥表現(xiàn)。 (2)支氣管壁厚度(WAt/Pi)、支氣管壁平滑肌厚度(WAm/Pi)、支氣管壁平滑肌細胞核數(shù)量(N/Pi)：哮喘組(A組)、低濃度LPS組(B組)及高濃度LPS組(C組)支氣管壁厚度(WAt/Pi)、支氣管壁平滑肌厚度(WAm/Pi)、支氣管壁平滑肌細胞核數(shù)量(N/Pi)均顯著高于正常對照組(D組)(P0.01)；低濃度LPS組(B組)支氣管壁厚度(WAt/Pi)、支氣管壁平滑肌厚度(WAm/Pi)、支氣管壁平滑肌細胞核數(shù)量(N/Pi)顯著高于哮喘組(A組)(P0.01),高濃度LPS組(C組)支氣管壁厚度(WAt/Pi)、支氣管壁平滑肌厚度(WAm/Pi)、支氣管壁平滑肌細胞核數(shù)量(N/Pi)顯著低于哮喘組(A組)(P0.01)。 (3)各組大鼠氣道阻力比較發(fā)現(xiàn)：哮喘組(A組)、低濃度LPS組(B組)、高濃度LPS組(C組)氣道阻力均明顯高于正常對照組(D組)(P0.01)；高濃度LPS組(C組)氣道阻力顯著低于哮喘組(A組)(P0.01),低濃度LPS組(B組)與哮喘組(A組)比較差異無統(tǒng)計學意義(P0.05)；高濃度LPS組(C組)氣道阻力明顯低于低濃度LPS組(B組)(P0.01)。 (4) RT-PCR檢測各組哮喘大鼠氣道平滑肌中TLRmRNA表達水平：哮喘組(A組)、低濃度LPS組(B組)、高濃度LPS組(C組)明顯高于正常對照組(D組)(P0.01)；低濃度LPS組(B組)、高濃度LPS組(C組)氣道平滑肌TLR4mRNA表達水平明顯高于哮喘組(A組)(P0.01)；高濃度LPS組(C組)氣道平滑肌TLR4mRNA表達水平明顯高于低濃度LPS組(B組)(P0.05)。 結(jié)論： (1)LPS通過激活哮喘大鼠ASMCs表面TLR4,使ASMCs合成分泌Th1/Th2型細胞因子失衡,從而參與哮喘氣道炎癥改變。 (2)TLR4在哮喘氣道炎癥及氣道重塑中起重要作用,LPS通過激活TLR4在哮喘氣道炎癥及氣道重塑的發(fā)病過程中可能發(fā)揮雙向調(diào)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.
【學位授予單位】：桂林醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R562.25
本文編號：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.
【學位授予單位】：桂林醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R562.25
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相關(guān)期刊論文 前3條
1 韋江紅;莫碧文;黃劍偉;徐青;林武州;;TOLL樣受體4對哮喘氣道平滑肌細胞分泌IL-5、IL-8的影響[J];山東醫(yī)藥;2011年03期
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