【作者】 王秀丹；

【导师】 张建勇；

【作者基本信息】 遵义医学院 ， 内科学， 2010， 硕士

【摘要】 第一部分哮喘小鼠气道黏液分泌的变化目的观察哮喘小鼠气道炎症的表现,并证实哮喘小鼠气道黏液高分泌的存在。方法将20只清洁级BALB/c小鼠随机分为2组：哮喘组(OVA组)、对照组(NS组),每组10只。哮喘组用卵蛋白(OVA)致敏和激发制作哮喘模型,对照组用生理盐水致敏和激发。检测支气管肺泡灌洗液(BALF)中细胞总数和细胞分类计数,采用酶联免疫吸附试验(ELISA)检测BALF中的IL-4和肿瘤坏死因子-α(TNF-α)水平,用阿尔辛蓝—过碘酸雪夫氏染色(AB-PAS)气道杯状细胞、用免疫组织化学检测气道组织中Muc5ac蛋白的表达及实时荧光定量逆转录聚合酶(RT-PCR)检测Muc5ac mRNA在气道组织的表达。结果OVA组小鼠BALF中的细胞总数、淋巴细胞和嗜酸性粒细胞高于NS组小鼠(P均<0.01),BALF中IL-4和TNF-α水平、气道杯状细胞阳性着色面积及气道组织中Muc5ac蛋白及其mRNA较NS组升高(P均<0.01)。结论哮喘小鼠存在气道炎症及气道黏液高分泌。第二部分TGF-β1/Smad信号通路在哮喘小鼠气道黏液高分泌中的作用及吡非尼酮的干预影响目的观察TGF-β1/Smad信号通路在支气管哮喘小鼠气道黏液高分泌中的作用。方法清洁级BALB/c小鼠40只,随机分为5组：正常对照组(NS组)8只,哮喘组(OVA组)8只,地塞米松干预组(OVA+DEX组)8只,溶剂对照组(OVA+SJ组)8只,吡非尼酮干预组(OVA+PFD组)8只。测定BALF中细胞总数和细胞分类计数,采用酶联免疫吸附试验(ELISA)检测BALF中的IL-4和TNF-a水平,用阿尔辛蓝—过碘酸雪夫氏染色(AB-PAS)对气道杯状细胞进行染色,用免疫组织化学法检测气道组织Muc5ac和肺组织TGF-βl的表达及实时荧光定量逆转录聚合酶(RT-PCR)检测Muc5ac mRNA、TGF-β1mRNA、Smad3及Smad4在肺组织内的表达。结果OVA组在BALF细胞总数、嗜酸性粒细胞数及气道杯状细胞阳染面积及免疫组化黏蛋白Muc5ac、转化生长因子TGF-β1积分光密度值及肺组织Muc5ac mRNA、TGF-p1mRNA、Smad3 mRNA、Smad4 mRNA表达与OVA+PFD组、NS组比较差异有统计学意义(P均<0.01), OVA组在BALF细胞总数、嗜酸性粒细胞数及AB-PAS阳染面积及免疫组化黏蛋白Muc5ac与OVA+DEX组比较差异有统计学意义(P均<0.01),但肺组织TGF-p1积分光密度值及TGF-β1 mRNA、Smad3 mRNA、Smad4 mRNA的表达水平两组无差异性(P>0.05)。OVA+PFD组小鼠气道杯状细胞及气道组织中的Muc5ac蛋白和TGF-β1及Muc5ac mRNA、TGF-β1 mRNA, Smad3 mRNA、Smad4 mRNA低于OVA+DEX组(P均＜0.01)。OVA组和OVA+SJ组在上述指标则组间差异无统计学意义(P>0.05)。结论哮喘小鼠肺组织TGF-β1蛋白、Smad3、Smad4蛋白及其mRNA和气道组织Muc5ac蛋白及其mRNA均成高表达改变。吡非尼酮可以抑制肺组织TGF-β1及Smad蛋白的表达,下调气道组织黏蛋白Muc5ac的表达,提示TGF-β1/Smad信号通路在气道黏液高分泌中发挥作用。更多还原

【Abstract】 Part 1 The airway mucus hypersecretion in asthmatic miceObjective To investigate airway inflammation in asthmatic mice, and confirm that asthmatic mice have mucus hypersecretion. Method Twenty clean BALB/c rats were randomly divided into two groups:control group asthmatic model group(OVA group, n=10), (NS group, n=10). asthmatic model group made asthmatic model in sensitized and challenged with ovalbumin (OVA), control group was sensitized and challenged with normal sodium. After three weeks, total cells and differential inflammatory cells were counted in bronchoalveolar lavage fluid (BALF), the levels IL-4 and TNF-a in BALF were determined by enzyme-linked immuno sorbent assay (ELISA), positive staining in the area of the goblet cell in airway wall was observed by a first blue-periodic acid Schiff reagent staining (AB-PAS), the expression of Muc5ac in airway were observed by immunohistochemical staining, the expressions of Muc5ac mRNA in airway tissue were observed real-time quantitative reverse transcription polymerase reaction (RT-PCR). Result Total cells, lymphocyte and eosinophil cells in BALF of athmatic model group were obviously higher than control group(P<0.01); the levels of IL-4 and TNF-a in BALF, positive staining in the area of the goblet cell in airway wall, and Muc5ac in the protein and gene level were higher than those in control group(P<0.01). Conclusion asthmatic mice have apparente inflammatory cell infiltration, and secrete a great quantity mucosubstance.Pary 2 The effect of TGF-β1/Smad signaling pathways in airwaymucus hypersecretion of asthmatic mice treated with pirfenidone Objective To investigate the effect of TGF-β1/Smad signaling pathways on mucus of hypersecretion asthmatic mice treated with pirfenidone.Methods Forty clean BALB/c rats were randomly divided into five groups:control group (NS group, n=8), asthmatic model group(OVA group, n=8), dexamethasone (DEX) treatment group(OVA+DEX group, n=8)),pirfenidone (PFD) treatment group (OVA+PFD group, n=8), Solvent control group (OVA+SJ group,n=8).Total cells and differential inflammatory cells were counted in bronchoalveolar lavage fluid (BALF), the levels of IL-4 and TNF-a in BALF were determined by enzyme-linked immuno sorbent assay (ELISA), positive staining in the area of the goblet cell in airway wall was observed by alcian blue/periodic acid schiff staining (AB-PAS), the expressions of Muc5ac in airway tissue and TGF-β1 in lung tissue were observed by immuneohistochemical staining, the expressions of Muc5acmRNA and TGF-β1mRNA and Smad3 mRNA and Smad4 mRNA in lung tissue were observed by real-time quantitative reverse transcription polymerase reaction (RT-PCR).Result Total cells、eosinophil cells and lymphocyte cells in BALF, the levels of IL-4 and TNF-a in BALF, positive staining in the area of the goblet cell in airway wall, and the expression of Muc5ac in the protein and gene level of OVA group were obviously higher than those in control group and (OVA+PFD) group (P<0.01), TGF-β1, Smad3 and Smad4 in the protein and gene level were higher than those in control group and (OVA+PFD) group (P<0.01), while were Non-significance difference Between OVA group and (OVA+DEX) control group(P>0.05).Conclusion The expressions of TGF-β1、Smad3 and Smad4 in the protein and gene level in lung tissue were higher than those in control group,and Muc5ac in the protein and gene level in airway tissue were higher than those in control group(P<0.01) PFD may inhibit the expressions of TGF-β1、Smad3 and Smad4 in the protein and gene level in lung tissue, and down the expressions of Muc5ac in the protein and gene level in airway tissue, illustrating TGF-β1/Smad signal pathway play a role in airway mucus hypersecretion.更多还原