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喘可治注射液对支气管哮喘免疫调控作用的临床与实验研究
Clinical and Experimental Study on the Immune Regulation Effect of Chuankezhi Injection in Asthma
【作者】 刘文华；
【导师】 黄东晖；
【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2011， 硕士
【摘要】 研究目的以哮喘免疫治疗的研究热点——Foxp3+ CD4+CD25+调节性T细胞的诱导为切入点,通过临床和实验两方面研究,探讨补肾温阳模式中药(喘可治注射液)在哮喘免疫调控中的作用及其机制。研究方法1.实验研究将Balb/C小鼠随机分为3组：对照组、哮喘组、喘可治(CKZ)组。采取以OVA经腹腔注射致敏加滴鼻激发复制哮喘模型,喘可治组给予喘可治注射液腹腔注射治疗,对照组与哮喘组给予生理盐水腹腔注射。分别观察各组小鼠的肺部病理改变,检测支气管肺泡灌洗液(BALF)中EOS情况；流式细胞术检测脾淋巴细胞中Foxp3+CD4+CD25+T细胞/CD4+T细胞比例；免疫组化检测肺组织Foxp3蛋白的细胞表达情况；ELISA检测脾淋巴细胞上清中IL-4、IL-10和TNF-β水平。2.临床研究选取我院呼吸内科50例哮喘患者,随机分为对照组和观察组(CKZ组),对照组给予常规治疗,观察组在常规治疗基础上加予喘可治注射液肌注足三里(4m1/次,qd),疗程为7天。观测并记录治疗期间主症、PEF、总IgE、EOS、外周血T淋巴细胞亚群的变化。研究结果(一)实验部分1.病理方面：喘可治注射液可降低哮喘小鼠气道周围炎症细胞尤其是EOS的浸润,减轻气道周围平滑肌增生。2.流式细胞术检测方面：哮喘小鼠脾淋巴细胞中Foxp3+CD4+CD25+T细胞/CD4+T细胞比例低于对照组,喘可治注射液可显著提高此细胞比例。3.免疫组化检测方面：哮喘小鼠肺组织Foxp3蛋白的表达低于对照组,喘可治注射液明显提高Foxp3的表达。4.ELISA检测方面：哮喘小鼠脾淋巴细胞上清中存在高IL-4,低IL-10、TNF-β的失衡状态,喘可治注射液可纠正这种失衡的极化状态。(二)临床部分1.疗效方面：喘可治组综合疗效优于对照组,喘可治注射液对寒哮证和虚哮证的疗效优于热哮证,差异有统计学意义(P<0.05)。2.实验室检查指标方面：喘可治注射液可降低哮喘患者外周血EOS及IgE水平,与对照组比较均有统计学差异；治疗前后PEF改善程度与对照组相近。3.实验室检查指标方面：喘可治注射液治疗后,与对照组相比CD4+细胞明显减少,CD8+细胞显著增多,两组相比有统计学意义(P<0.05)研究结论1.哮喘存在Foxp3+CD4+CD25+调节性T细胞相对数量不足和功能缺陷,Thl和Th2之间的失衡参与了哮喘的发病机制；2.喘可治注射液能有效减轻哮喘气道的变态反应炎症；3.以喘可治注射液为代表的补肾温阳中药能抑制Th2细胞的增殖,促进Foxp3+CD4+CD25+调节性T细胞的活化和关健的转录因子Foxp3表达的增加而产生免疫耐受机制,抑制了炎症反应；4.抑制性细胞因子IL-10和TNF-β可能参与了喘可治注射液诱导的Foxp3+CD4+CD25+调节性T细胞免疫抑制作用的机制。
【Abstract】 ObjectiveAim to investigate the immune regulation mechanism of Chuankezhi injection(CKZ) which is a Chinese medical model formula of reinforcing kidney and warming yang in the managment of bronchial asthma.Methods1.Animal Experiment:Mice were sensitized and challenged with ovalbumin (OVA) to establish allergic asthma-like model. To determine the contribution of Chuankezhi injection on airway allergic inflammation and immune regulation, we examined the BALF cell differentiated count, And lung histopathology, proportion of spleen Foxp3+CD4+CD25+Treg cells/CD4+T cells were measured by flowcytometry. Cytokines IL-4、IL-10 and TNF-βlevels in the cultured splenocytes supernatant were examined with ELISA. Foxp3 expression in lung was analyzed with immunohistochemistry.2. Clinical study:50 mild or moderate asthmatic patients collected from the department of Respiratory Medicine, Guangdong Provincial hospital of TCM were randomized into control group and Chuankezhi (CKZ) group. The control group received conventional treatment. CKZ group received the intramuscular injection (4ml qd x 7days) and the conventional treatment as well. The changes of the primary symptoms、PEF values、IgE level, blood T cell subsets were recorded before and after the treatment.Results1. Animal Experiment Administration of Chuankezhi injection prevented the eosinophil accumulation in BALF in the lungs of asthma. CKZ group mice have a lower spleen Foxp3+CD4+CD25+Treg cells/CD4+T cells proportion compared with control group. Asthma group mice had higher IL-4 and lower IL-10、TNF-βlevels in the cultured splenocytes supernatant than control group. And Chuankezhi injection corrected the polarization respectively. Foxp3 expression in asthma mice lung was significantly lower than that in control group. And there was a higher level of Foxp3 due to the treatment with Chuankezhi injection.2. Clinical study:The curative effects in 2 groups were significant (p<0.05) by statistical analysis, the effect in CKZ group was better than that in control group.Except for the major symptoms, CKZ group can lower EOS and IgE lever, correct the imbalance of CD4+ and CD8+ when compared with control group.2 groups can improve the peak expiratory flow rates. The comparison of PEFR after treatment were significant (p<0.05) by statistical analysis in the two group.Conclusions1. Insufficiency and dysfunction of Foxp3+CD4+CD25+Treg cell, as well as the imbalance between Thl and Th2 lymphocytes may play an important role in the pathogenesis of asthma.2. Chuankezhi injection can alleviate allergic inflammation in the airway in asthma patients.3. Chuankezhi injection inhibit the proliferation of Th2, and promote the activation of the Foxp3+CD4+CD25+ Treg cell and up-regulate the expression of the master transcription factor foxp3 to conduct immunotolerance.4. Regulatory cytokines IL-10, TNF-βmay be involved in the regulatory mechanisms of Foxp3+CD4+CD25+Treg cells induced by Chuankezhi injection.