【作者】 白枫；

【导师】 黎海芪；

【作者基本信息】 重庆医科大学 ， 儿科学， 2009， 博士

【摘要】 第一部分金银花有效成分筛选及对卵清蛋白致敏小鼠的抗过敏作用【目的】观察金银花中绿原酸、木犀草素和黄褐毛忍冬总皂苷的抗食物过敏作用。【方法】建立雌性BALB/c小鼠食物过敏模型,分别以高、中、低浓度的绿原酸、木犀草素、黄褐毛忍冬总皂苷作用于过敏小鼠。采用ELISA法测定小鼠血清中OVA特异性IgE水平,以足垫肿胀实验检测迟发型超敏反应(DTH),筛选出金银花中抗食物过敏的有效成分。进一步检测该有效成分对空肠绒毛形态(HE染色)、空肠和肠系膜肥大细胞(甲苯胺蓝染色)以及小肠黏液中总IgA及OVA特异性IgA水平的影响。【结果】绿原酸、木犀草素及中、低浓度的黄褐毛忍冬总皂苷用于食物过敏小鼠模型后对足垫肿胀反应及血清中OVA特异性IgE未产生抑制作用。高浓度黄褐毛忍冬总皂苷可抑制小鼠足垫肿胀反应,降低血清中OVA特异性IgE水平,缓解肠道及肠系膜中肥大细胞的聚集和脱颗粒现象,减轻小肠绒毛炎症,但对小肠黏液总IgA和OVA特异性IgA无明显影响。【结论】绿原酸、木犀草素对食物过敏无明显治疗作用。高浓度Ful可同时缓解食物过敏小鼠IgE介导的及细胞介导的超敏反应。第二部分黄褐毛忍冬总皂苷对卵清蛋白致敏小鼠脾脏T细胞功能亚群的影响【目的】观察黄褐毛忍冬总皂苷对卵清蛋白致敏小鼠脾脏T细胞功能亚群的影响,以探讨Ful的抗过敏机制。【方法】建立雌性BALB/c小鼠食物过敏模型。取小鼠24只,均分为3组,即食物过敏组(FA)、药物干预组(Ful)和生理盐水对照组(NS)。Ful组小鼠自第20日起每日皮下注射Ful 200 mg/kg,共22 d。小鼠脾脏单个核细胞中加入OVA培养48小时。ELISA法检测各组小鼠血清中OVA特异性IgE水平;流式细胞术检测脾脏单个核细胞中CD4+CD25+Foxp3+调节性T细胞占CD4+ T细胞比例;ELISA法检测脾脏单个核细胞培养上清中IL-4、IFN-γ、IL-10水平。【结果】食物过敏小鼠血清中OVA特异性IgE水平增高,脾脏CD4+CD25+Foxp3+ T细胞占CD4+ T细胞比例与NS组无差别,脾脏单个核细胞培养上清中IL-4分泌增加,IFN-γ的产生与NS组无差别,IFN-γ/IL-4低于NS组。Ful干预后过敏小鼠OVA特异性IgE水平降低,CD4+CD25+Foxp3+ T细胞占CD4+ T细胞比例明显增高,IL-4水平降低,IFN-γ/IL-4升高。IL-10产生水平各组间差异无统计学意义。【结论】黄褐毛忍冬总皂苷可诱导OVA致敏小鼠脾脏中CD4+CD25+Foxp3+ T细胞,增强Treg反应,并相对削弱Th2反应,从而对IgE介导的超敏反应起到缓解作用。第三部分黄褐毛忍冬总皂苷对卵清蛋白致敏小鼠肠道炎症因子和抗炎因子的影响【目的】观察黄褐毛忍冬总皂苷对卵清蛋白致敏小鼠肠道炎症因子和抗炎因子的影响。【方法】建立雌性BALB/c小鼠食物过敏模型。取小鼠24只,均分为3组,即食物过敏组(FA)、药物干预组(Ful)和生理盐水对照组(NS)。Ful组小鼠自第20日起每日皮下注射Ful 200 mg/kg,共22 d。采用RT-PCR法检测小鼠空肠组织中TGF-β1、IL-6、IL-17A、Foxp3、IL-10 mRNA表达;免疫组织化学法检测小鼠空肠组织中TGF-β1、IL-6、IL-17A蛋白表达;检测空肠中髓过氧化物酶(MPO)活性代表中性粒细胞活化水平。【结果】食物过敏小鼠空肠中TGF-β1、IL-6、IL-17A的mRNA和蛋白表达水平均增高,而Foxp3 mRNA表达下降。经Ful干预后,小鼠TGF-β1表达未下降,但IL-6、IL-17A表达水平均明显降低,Foxp3 mRNA表达明显增高。IL-10 mRNA表达、空肠组织中髓过氧化物酶水平各组间比较差异无统计学意义。【结论】食物过敏发生时肠道内存在以IL-6、IL-17A表达增高为主的炎症反应。黄褐毛忍冬总皂苷可有效降低OVA致敏小鼠肠道炎症因子IL-6、IL-17A的过度表达,并显著增强CD4+CD25+调节性T细胞特异性转录因子Foxp3的表达,这可能是黄褐毛忍冬总皂苷改善肠道炎性病理改变的机理之一。第四部分黄褐毛忍冬总皂苷对卵清蛋白致敏小鼠肺部免疫状态的影响【目的】观察黄褐毛忍冬总皂苷对OVA致敏BALB/c小鼠肺部免疫状态的影响。【方法】取BALB/c雌鼠96只,均分为6组:食物过敏+PBS雾化吸入组(FA+AI-PBS)、食物过敏+OVA雾化吸入组(FA+AI-OVA)、食物过敏+PBS雾化吸入+Ful干预组(FA+AI-PBS+Ful)、食物过敏+OVA雾化吸入+Ful干预组(FA+AI-OVA+Ful)、单纯致敏+OVA雾化吸入组(Se+AI-OVA)及生理盐水对照组(Control)。FA+AI-PBS和FA+AI-OVA组小鼠经OVA致敏及灌胃激发,d41和d42分别给予PBS和OVA雾化吸入。Se+AI-OVA组未经OVA灌胃激发,其余处理同FA+AI-OVA组。FA+AI-PBS+Ful组及FA+AI-OVA+Ful组给予高浓度Ful(200 mg/kg)皮下注射治疗共22 d,其余处理分别同FA+AI-PBS和FA+AI-OVA组。瑞氏-姬姆萨复合染色观察支气管肺泡灌洗液(BALF)中白细胞数及分类;肺组织HE染色进行气道炎症病理分析;免疫组织化学法观察肺组织中TGF-β1、IL-6、IL-17A蛋白表达水平;检测肺组织中髓过氧化物酶活性代表中性粒细胞活化水平。【结果】FA+AI-PBS组小鼠支气管肺泡灌洗液中细胞总数与分类计数与对照组无差异,肺部亦无明显的炎细胞浸润;FA+AI-OVA组小鼠BALF中细胞总数增加,中性粒细胞、淋巴细胞百分比显著增高,镜下可见大量红细胞,肺组织炎症病理评分以及肺组织中TGF-β1、IL-6、IL-17A表达水平均明显增高。Se+AI-OVA组小鼠肺组织炎症病理评分低于FA+AI-OVA组,且肺部以嗜酸粒细胞浸润为主,肺部TGF-β1、IL-6、IL-17A表达低于FA+AI-OVA组。黄褐毛忍冬总皂苷干预后吸入OVA的食物过敏小鼠肺部TGF-β1表达水平无明显改变,炎症因子IL-6、IL-17A的表达降低,但尚未能有效减轻中性粒细胞在局部的聚集及改善肺部炎症反应。【结论】食物过敏小鼠吸入特异性抗原后,可导致肺组织中TGF-β1、IL-6、IL-17A表达明显增高,引起严重的以中性粒细胞浸润为主的炎症。黄褐毛忍冬总皂苷可在一定程度上降低吸入OVA的食物过敏小鼠肺组织中炎症因子IL-6、IL-17A的表达,但尚未能明显改善肺部病理状态。更多还原

【Abstract】 Part one Screening for effective ingredients in lonicera japonica which have anti-allergic effects on ovalbumin-sensitized BALB/c mice[Objective] To investigate anti-allergic effects of chlorogenic acid, Luteolin and fulvotomentoside (Ful) on ovalbumin-sensitized BALB/c mice.[Methods] Female BALB/c mice were sensitized and challenged with OVA. The mice were treated with chlorogenic acid, Luteolin and Ful of high, medium and low concentration, respectively. To screen effective ingredients in Lonicera japonica which had anti-allergic effects, the levels of OVA-specific IgE in serum were measured by ELISA and the footpad swelling reaction was assessed for the OVA-induced delayed hypersensitivity. More detections were done to explore the effects of the effective ingredient(s). Histological examinations of jejunum were performed by HE staining and the mast cells in jejunum and mesentery were observed by toluidine blue staining. The levels of total IgA and OVA-specific IgA in the intestinal mucus were measured by ELISA.[Results] Chlorogenic acid, luteolin and medium or low concentration of Ful had no inhibitory effects on footpad swelling reaction and OVA-specific IgE in serum. The footpad swelling reaction was significantly inhibited and the levels of OVA-specific IgE in serum were reduced after giving high concentration of Ful. Moreover, high concentration of Ful could inhibit aggregation and degranulation of mast cells in jejunum and mesentery and relieve inflammatory reactions in villi of small intestine, but no obvious effects on the levels of total IgA and OVA-sIgA in the intestinal mucus.[Conclusions] Chlorogenic acid and luteolin had no significant therapeutic effects on BALB/c mice with food allergy. High concentration of Ful may be of potential research value in treatment for both IgE and non-IgE mediated food allergy.Part two Effects of fulvotomentoside on splenic T lymphocyte subsets of ovalbumin-sensitized BALB/c mice[Objective] To investigate the effects of Ful on the splenic T lymphocyte subsets of OVA-sensitized BALB/c mice. [Methods] Female BALB/c mice were randomly divided into 3 groups, group food allergy (FA), group Ful and group normal sodium (NS). Mice in group FA and group Ful were sensitized and challenged with OVA. Mice in group Ful were treated with 200 mg/kg concentration of Ful daily for 22 d. The levels of OVA-specific IgE in serum were measured by ELISA. Mononuclear cells in spleen were cultured with OVA for 48 h. Flow cytometry was used to detect the percentage of CD4+ CD25+ Foxp3+ regulatory T cells in CD4+ T cells in mononuclear cells in spleen. The levels of IL-4, IFN-γand IL-10 in the supernatants were detected by ELISA.[Results] Compared with group NS, the levels of OVA-specific IgE and IL-4 were increased and the ratio of IFN-γ/IL-4 was decreased in group FA. There was no difference of the percentage of CD4+ CD25+ Foxp3+ regulatory T cells between group FA and group NS. The levels of OVA-specific IgE and IL-4 were reduced after the treatment with Ful. The percentage of CD4+ CD25+ Foxp3+ regulatory T cells and the ratio of IFN-γ/IL-4 were increased after giving Ful. No difference was found of the levels of IL-10 among the three groups.[Conclusions] Ful could induce CD4+ CD25+ Foxp3+ regulatory T cells and weaken Th2 response in spleen. Ful may be of value in the treatment for IgE-mediated food allergy. Part three Effects of fulvotomentoside on inflammatory factors and antiinflammatory factors in intestine of ovalbumin-sensitized BALB/c mice[Objective] To investigate the effects of fulvotomentoside on inflammatory factors and antiinflammatory factors in intestine of ovalbumin-sensitized BALB/c mice.[Methods] Female BALB/c mice were randomly divided into 3 groups, group food allergy (FA), group Ful and group normal sodium (NS). Mice in group FA and group Ful were sensitized and challenged with OVA. Mice in group Ful were treated with 200 mg/kg concentration of Ful daily for 22 d. The mRNA expressions of TGF-β1, IL-6, IL-17A, Foxp3 and IL-10 in jejunum were detected by RT-PCR. The protein expressions of TGF-β1, IL-6, IL-17A in jejunum were detected by immunohistochemical method. The activation of neutrophils in jejunum was assayed by the level of MPO.[Results] Compared with group NS, the expressions of TGF-β1, IL-6, IL-17A mRNA and protein in jejunum were increased and the Foxp3 mRNA expression was decreased in group FA. After the treatment with Ful, IL-6 and IL-17A expressions were decreased and foxp3 mRNA expression was increased, and no change of TGF-β1 expression was found. There were no differences of IL-10 mRNA expression and the level of MPO among the three groups.[Conclusions] Inflammatory reaction which was characteristed by the increase of IL-6 and IL-17A expressions was found in intestine of ovalbumin-sensitized BALB/c mice. Ful could decrease overexpression of IL-6 and IL-17A, and increase the expression of specific transcription factor Foxp3 of CD4+ CD25+ regulatory T cells significantly in intestine. This may be one of the mechanisms that Ful improved intestinal inflammation.Part four Effects of fulvotomentoside on immune state in lung of ovalbumin-sensitized BALB/c mice[Objective] To investigate the effects of fulvotomentoside on immune state in lung of ovalbumin-sensitized BALB/c mice.[Methods] Ninety-six female BALB/c mice were randomly divided into 6 groups, group FA+AI-PBS, group FA+AI-OVA, group FA+AI-PBS+Ful, group FA+AI-OVA+Ful, group Se+AI-OVA and group Control. Mice in group FA+AI-PBS and group FA+AI-OVA were sensitized intraperitoneally and challenged intragastrically with OVA. Mice in Group FA+AI-OVA and group Se+AI-OVA were exposed to OVA by atomizing inhalation on d41 and d42. Mice in group Se+AI-OVA were sensitized intraperitoneally but not challenged intragastrically with OVA. Mice in group FA+AI-PBS+Ful and group FA+AI-OVA+Ful were treated with Ful, Other processes were the same as the mice in group FA+AI-PBS and group FA+AI-OVA, respectively. The number of total leukocytes and cell classification in bronchoalveolar lavage (BALF) were counted, and inflammatory characteristic of lung was scored by staining with hematoxylin and eosin. The protein expressions of TGF-β1, IL-6, IL-17A in lung of the mice were detected by immunohistochemical method. The activation of neutrophils in lung was assayed by the level of MPO.[Results] No difference was found of number of total leukocytes and cell classification in BALF between group FA+AI-PBS and group control. There was no inflammatory cells infiltration in lung of the mice in group FA+AI-PBS. Numbers of total leukocytes and erythrocytes as well as the percentage of neutrophils and lymphocytes were increased in group FA+AI-OVA. Inflammatory score and protein expressions of TGF-β1, IL-6, IL-17A in lung were increased, too. Inflammatory score and TGF-β1, IL-6, IL-17A expressions in group Se+AI-OVA were lower than those in group FA+AI-OVA. Eosinophils infiltration was significant in group Se+AI-OVA. After the treatment with Ful, TGF-β1 expression did not change and IL-6, IL-17A expressions were decreased in lung of the mice that inhaled OVA. It was not enough for Ful to relieve the neutrophil aggregation and improve inflammatory reaction in lung.[Conclusions] The expressions of TGF-β1, IL-6, IL-17A in lung of the mice with food allergy were increased markedly after they inhaled specific antigen, which caused serious inflammation that was induced by neutrophil infiltration in lung. Ful could decreased the expressions of IL-6, IL-17A to some extent, but it was not enough to improve pathologic state in lung.更多还原