【作者】 陈佳；

【导师】 赵国平；

【作者基本信息】 暨南大学 ， 中西医结合临床， 2010， 硕士

【摘要】 目的：建立嗜酸细胞性胃肠炎(EG)小鼠模型,用半夏泻心汤干预治疗,通过相关药效学指标验证该模型是否可以作为半夏泻心汤方证模型；检测胃肠粘膜主要碱性蛋白(MBP) mRNA表达水平,探讨半夏泻心汤是否通过作用于毒覃碱受体这一机制调节胃肠运动,治疗EG。方法：实验用雌性Balb/c小鼠34只,适应性喂养一周后,随机分为2组,空白对照组8只,处理组26只。处理组以腹腔注射OVA/Al(OH)3结合PLGA包裹的OVA颗粒灌胃制备EG小鼠模型。观察胃肠道病理切片,检测外周血嗜酸性粒细胞,IgE、IgG1含量,以验证造模成功。将处理组随机分为治疗组、西药对照组、中药对照组、模型组。治疗组给予半夏泻心汤,中药对照组给予香砂六君子汤,西药对照组给予泼尼松龙治疗,模型组给予等量生理盐水。每天给药一次,共20天,给药结束后第三天取材。HE染色观察实验各组小鼠胃肠粘膜组织病理学变化。采用酶联免疫技术测定小鼠外周血IgE、IgG1含量,荧光定量PCR技术测定小鼠胃肠组织中MBP mRNA表达。采用统计学软件SPSS13.0进行分析。结果：1.EG小鼠一般情况观察：空白对照组小鼠毛发光泽,精神好,食量和便质正常,体重增长明显。与空白对照组相比,模型组小鼠,毛色枯黄、精神差、饮食减少、腹泻、便质稀软,体重增长不明显。西药对照组和半夏泻心汤组,体重较用药前增加明显,毛色较之前有光泽、精神好转、便质较稠,有所改善。与半夏泻心汤组相比,中药对照组小鼠一般情况虽有所改善,但不如其疗效明显。2.EG小鼠病理形态的观察：空白对照组小鼠胃肠粘膜结构层次清楚,无炎症、充血与水肿；模型组与空白对照组相比,可见胃肠粘膜组织缺损,充血,水肿,糜烂,有中性粒细胞、嗜酸性粒细胞炎症细胞浸润。西药对照组与半夏泻心汤组炎症部位较小,粘膜层增厚,充血,水肿,糜烂较模型组轻,有肉芽组织生成覆盖溃疡面,炎症细胞浸润较少。中药对照组炎症反应较半夏泻心汤组重,仍可见大量炎症细胞浸润。3.外周血中嗜酸粒细胞,IgE、IgG1含量的测定：造模完成后,处理组外周血嗜酸粒细胞,IgE、IgG1含量均比空白对照组高,差别具有统计学意义(P<0.05)。用药治疗后,模型组嗜酸粒细胞,IgE和IgG1含量与空白对照组相比,有显著性差异(P<0.05)；半夏泻心汤组、西药对照组,中药对照组与模型组相比,嗜酸粒细胞,IgE和IgG1含量降低,有显著性差异(P<0.05)；半夏泻心汤组与西药对照组相比,嗜酸粒细胞,IgE和IgG1含量的差别无统计学意义(P>0.05)；而中药对照组与半夏泻心汤组相比,嗜酸粒细胞含量的差别有统计学意义(P<0.05)；IgE和IgG1含量的差别无统计学意义(P>0.05),但半夏泻心汤组IgE和IgG1含量低于中药对照组的含量。4.胃肠组织中MBP mRNA表达水平测定：与空白对照组相比,模型组MBP mRNA表达水平增高,有显著性差异(P<0.05)；用药治疗后,西药对照组、半夏泻心汤组胃肠组织中MBP mRNA表达水平比模型组低,差别具有统计学意义(P<0.05)；中药对照组胃部MBP mRNA表达水平与模型组相比,差别有统计学意义(P<0.05),但其肠部MBP mRNA表达水平与模型组相比,差别无统计学意义(P>0.05)；半夏泻心汤组与西药对照组相比,差别无统计学意义(P>0.05)；而中药对照组与半夏泻心汤组相比,差别有统计学意义(P<0.05)。结论：1.单用腹腔注射OVA/A1(OH)3凝胶和OVA溶液,胃肠致敏作用不明显,追加PLGA包裹的OVA微粒灌胃,可以成功建立EG小鼠模型。2.采用半夏泻心汤治疗EG小鼠模型,疗效满意,与西药特效药泼尼松龙相似,疗效好于香砂六君子汤,表明EG小鼠模型可以作为半夏泻心汤方证模型。3.应用半夏泻心汤可降低EG小鼠外周血IgE、IgG1含量,使胃肠组织黏膜受损情况得以减轻,说明半夏泻心汤能够通过调节免疫功能参与胃肠组织的修复。4.嗜酸性粒细胞通过释放MBP以诱发毒蕈碱性受体(M2)的功能障碍,引起平滑肌收缩,促使神经轴突坏死,降低胃蠕动和胃排空的功能。EG小鼠模型MBP呈高表达。半夏泻心汤能抑制EG小鼠模型MBP mRNA的表达,提示该方可抑制MBP的释放,阻断由此诱发的毒蕈碱性受体(M2)功能障碍,减轻炎症,促使胃肠功能的恢复。更多还原

【Abstract】 Objective:To establish the model of mouse with EG, verify this model successfully and match with syndrome of banxiaxiexin decotion. After the treatment, to detect expression of grastrointestinal mucosa MBP mRNA and study the curative effect and mechanism of banxiaxiexin decotion to prevent EG by the way of M2 receptor.Methods:Female Balb/c mice 34, were randomly divided into 2 groups, normal group 8, treatment group 26. The mice were intraperitoneal injected OVA/Al(OH)3 and lavaged with PLGA microparticles containing OVA modeling. We should fetch blood from mice eye department, detect the peripheral blood eosinophils, IgE and IgGl content to validate the model successfully. The same time, treatment group were randomly divided into model group, banxiaxiexin decotion group, xiangshaliujunzi decotion and western medicine group. Those groups were administered once daily,20 days, to be drawn materials after the third day. The pathological changes in mouse gastrointestinal mucosa of experimental groups was observed by HE stain. The content of peripheral blood IgE and IgGl were determined by ELISA. The expression level of MBP mRNA in gastrointestinal tissues was determined by quantitative PCR. We used SPSS 13.0 statistical analysis software.Results:1. EG mice general state of health:Compared with normal group, model group mice, have yellow coat, the spirit of poor food intake, diarrhea and soft dejecta, the information of medicine group and banxiaxiexin decotion group has been improved, but the curative effect of xiangshaliujunzi decotion is less than banxiaxiexin decotion.2. EG mice pathological observation:In normal group the gastrointestinal mucosa layer is integrity, there are no gastric ulcer focus, hyperemia and edema. In model group, banxiaxiexin decotion group and western medicine group, it can be found different degree of gastrointestinal mucosal tissue defects, inflammatory disease, but the information has been improved, xiangshaliujunzi decotion group still has more inflammation and inflammatory cell.3. The content of peripheral blood eosinophils, IgE and IgGl to be determined:The level of peripheral blood eosinophils, IgE and IgGl in treatment group is higher than normal group (P＜0.05), the statistical analysis has significant difference. After drug treatment, blood eosinophils, IgE and IgGl contents in model group and normal group, there are significant differences (P＜0.05); banxiaxiexin decotion group, western medicine group and xiangshaliujunzi decotion group compared with model group, there are significant differences (P＜0.05); blood eosinophils, IgE and IgGl contents in banxiaxiexin decotion group and Western medicine group, there are no significant difference (P>0.05); while blood eosinophils in xiangshaliujunzi decotion group compared with banxiaxiexin decotion group, there are significant differences (P＜0.05); IgE and IgGl contents in xiangshaliujunzi decotion group compared with banxiaxiexin decotion group, the difference was not statistically significance (P>0.05), but banxiaxiexin decotion group IgE and IgGl were lower than the content of Xiangsha Liujunzi Decoction group.4. The expression level of MBP mRNA in gastrointestinal tissues to be measured:After drug treatment, the expression level of MBP mRNA in gastrointestinal tissues of model group and normal group, there are significant differences(P<0.05), banxiaxiexin decotion group and western medicine group compared with model group, there are significant differences (P＜0.05); banxiaxiexin decotion group and Western medicine group, there are no significant difference (P>0.05); while xiangshaliujunzi decotion group compared with banxiaxiexin decotion group, here are significant differences (P＜0.05).Conclusions:1. To establish and verify the model of mouse with EG, the model is mde by OVA/Al(OH)3 and PLGA microparticles containing OVA. After modeling, The model of mouse with EG can cause pathological changes in gastrointestinal mucosa, the peripheral blood level of eosinophils, IgE and IgGl in treatment group increased, it may indicate that the model is successful.2. By the result of comparison, we indicate that banxiaxiexin decotion can improve the general situation and gastrointestinal pathological situation, its efficacy is as well as western medicine group and better than xiangshaliujunzi decotion group. It indicates that this model matchs with syndrome of banxiaxiexin decotion.3. Banxiaxiexin decotion can effectively reduces the level of peripheral blood IgE, IgGl and improve pathological changes in gastrointestinal mucosa, it indicates that banxiaxiexin decotion can be able to regulate immune function involved in gastrointestinal tissue repairing.4. The pathogenesis of EG is related to the mechanism of MBP in gastrointestinal tissues, MBP mainly through muscarinic receptors play in banxiaxiexin decotion. Banxiaxiexin decotion can reduce inflammation and promote the recovery of gastrointestinal function by reducing the expression level of MBP mRNA in gastrointestinal tissues.更多还原