【作者】 贺小英；

【导师】 靳瑞；

【作者基本信息】 广州中医药大学 ， 针灸推拿学， 2006， 博士

【摘要】 一、研究背景、目的与意义 哮喘气道的重构是指广泛的气道结构改变,如气道壁的增厚、上皮层鳞状上皮细胞的化生,杯状细胞的增生,平滑肌的增生、肥厚,上皮下的纤维化等。气道壁的增厚以及气道平滑肌的增生、肥大等,导致了气道的狭窄、气流阻力的增加及气道高反应性。气道重构是气道反复损伤与修复的结果,是造成不可逆转的气道阻塞的重要原因,是导致难治性支气管哮喘临床疗效不佳的主要原因。针灸作为一种传统的中医疗法,在治疗支气管哮喘有显著的临床疗效。为探讨针灸对哮喘气道重构的可能干预机制,本研究从对气道重构干预作用的角度观察电针背六穴(靳瑞教授临床针灸疗法精华——靳三针常用组穴法之一)对哮喘模型气道重构的气道超微结构的改变及对转化生长因子-β(transforming growth factor-β,TGF-β)、白细胞介素-11(Interleukin 11,IL-11)、粒细胞-吞噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)、胰岛素样生长因子-1(insulin growth factor 1,IGF-1)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的影响,为针灸治疗哮喘及靳三针在临床上的推广运用提供理论依据。 二、研究方法 文献研究综述了中医对哮喘病因病机的认识及其中药、针灸治疗方法;综述了现代医学对哮喘气道重构的发病和调控机制的认识以及哮喘气道重构的药物治疗,在此基础上进行评述和展望。 实验研究选择豚鼠作为实验动物。将豚鼠随机分成4组:正常对照组、电针组、地塞米松组、模型组。造摸方法:第1天以10%(10g/100mL)卵清蛋白注射液腹腔注射1ml致敏,于第15天起隔天以1%(1g/100mL)卵蛋白溶液超声雾化;隔天激发1次,共6周。干预治疗采用电针背六穴(大杼、风门、肺俞)。在每次超声雾化激发前进行,留针15min,隔天1次,共6周。治疗结束后,光学显微镜下观察各组豚鼠支气管和肺组织的病理形态学改变、嗜酸粒细胞计数,并进行病理形态学测量;采用电子显微镜观察支气管和肺组织的超微结构;采用免疫组织化学法检测与气道重构相关的细胞因子TGF-β、GM-CSF、IGF-1和IL-11及MMP-9在各组支气管和肺组织中的蛋白表达。 三、结果 3.1 文献研究结果 文献研究表明,目前哮喘气道重构的机制尚未完全明了,但近年来的研究表明多更多还原

【Abstract】 1 Background, purpose and significance of the researchAirway remodeling is referred to the extensive changes of airway structure, involving the thickening of the airway wall, metaplasia of squamous cell in airway epithelial layer, proliferation of airway goblet cells and smooth muscle, fibrosis of airway epithelial layer and differentiation of mesenchymal cells, particularly myofibroblasts. The above changes lead to the obstruction of airway, increase of airflow resistance and airway hyperresponsiveness. Airway remodeling is the results of airway repeated damage and repairing, which is contributed to the refractory bronchial asthma. Acupuncture has an definite effect on bronchial asthma. To explore the therapeutic mechanism of acupuncture for bronchial asthma and to supply evidence for the expanding application of Jin’s triple needling therapy in treating asthma, the effects of electroacupuncture （EA） of back six points on airway ultrastructure, transforming growth factor-β （TGF-β）, interleukin 11（IL-11）, granulocyte-macrophage colony-stimulating factor （GM-CSF）, insulin growth factor 1 （ IGF-1）,matrix metalloproteinase-9（ MMP-9） in asthmatic Guinea pigs with airway remodeling.2 MethodsA literature review was carried out to summarize the TCM etiology and pathogenesis of asthma, as well as its treatment with traditional Chinese herbal medicine and acupuncture. Meanwhile, the pathogenesis of airway remodeling and its regulatory mechanism and drug treatment were also discussed.The experimental studies were performed on Guinea pigs. Forty-eight healthy Guinea pigs were randomized into 4 groups: normal control group, model control group, dexamethasone group and EA group. The model was established by sensitization with intraperitoneal injection of 10% ovalbumin （OVA） 1ml in the first day and challenge with nebulized inhalation of 1% OVA solution in the 15^th day, every other day for 6 weeks. Every time before challenge, the interventive treatment of EA of back six points （Dazhu,Fengmen, Feishu） was carried out. After treatment, the pathological changes, eosinophils （EOS） count and morphometric analysis in bronchi and lung were observed under light microscope, the ultrastructure of bronchi and lung was observed under electron microscope, and the protein expressions of TGF-B, IL-11,GM-CSF, IGF-1 and MMP-9 were detected by immunohistochemical method.3 Results3.1 The results of literature reviewThe results of literature review showed that the mechanism of airway remodeling is inadequate. Some articles reported that various cytokines, inflammatory medium and enzymes were involved with the airway remodeling. They activated the smooth muscle cells, myofibroblasts and EOS and caused the proliferation and atrophy of smooth muscle, and the fibrosis of epithelial layer. As for its treatment, there has no specific remedy until now except the early treatment with cortical hormone at the time of the confirmed diagnosis.3.2 The results of experimental studies3.2.1 The pathological changes in model group were as follows: focal chronic infiltration of inflammatory cells in submucouS. connective tissue of main bronchus, vascular congestion, hyperplasia of- glands, diffuse chronic infiltration of inflammatory cells in alveoli of lung, narrowed lumen, consolidation of alveoli, indicating that the model was established successfully. The pathological changes in dexamethasone group and EA group were much relieved. The result of eosinophils （EOS） count showed that the number of EOS was（30.27+2.86） in the model group, much higher that （1.09+0.27） in the normal group, and the difference was significant （P<0.01）;EOS count in dexamethasone group and EA group was decreased as compared with the model group, the difference being significant（P<0.01）;as compared with that in dexamethasone group, EOS count in EA group was higher （P<0.05） . The results of morphometric analysisof bronchi and lung showed that the thickness of airway intima and thickness of fiber structure in main airway of the model group was higher than those in the normal group （P<0.05）, and were decreased in dexamethasone group and EA group as compared with the model group （P<0.05）;in small airway, the thickness of smooth muscle in the normal group was higher than those in the normal group （P<0.05）, and were decreased in dexamethasone group and EA group as compared with the model group （P<0.05）. The difference of the above three parameters was insignificant between dexamethasone group and EA group （P<0.05）.3.2.2 The changes of the ultrastructure of bronchi and lung under electron microscope inmodel group were as follows: injured pili of bronchial epithelial cells disarranged and became loose and short, microtubule in the pili was obstructed, some pili were dripped off;microvilli became less;columnar epithelial cell degenerated, with swollen chondrioid and chondrioid vacuolization and capsular endoplasmic reticulum;the intercelular space of base cells and columnar epithelial cells became wide with infiltration of a large amount of EOS;some EOS were activated and became low-density EOS after degranulation;cell desmosomes junctions were damaged and lumen formed with swollen nuclear membrane and widened nuclear membrane space;basilar membrane were unconnected with increaed collagen fiber and fibroblasts. The changes of ultrastructure of bronchi and lung in dexamethasone group and EA group were much relieved.3.2.3 The immunohistochemical results showed that the protein expressions of TGF-B, GM-CSF and IGF-I in bronchi and lung were higher in the model group than those in the normal group （P<0.01）, and were decreased in dexamethasone group and EA group as compared with the model group, the difference being significant （P<0.05）. The protein expressions of IL-11 and MMP-9 in bronchi and lung were higher in the model group than those in the normal group （P<0.01）, and were decreased in dexamethasone group and EA group as compared with the model group, the difference being insignificant （P>0.05）.4 ConclusionThe interventive treatment of EA of back six points （Dazhu, Fengmen, Feishu） can reduce EOS count in bronchi and lung of asthmatic guinea pigs, relieve the inflammatory reaction, decrease the thickness of airway intima and thickness of fiber structure in main airway and the thickness of smooth muscle in small airway to relieve the damage of ultrastructure. EA of back six points （Dazhu, Fengmen, Feishu） can decrease the protein expressions of TGF-8, GM-CSF and IGF-I, but has no obvious effect on the protein expressions of IL-11 and MMP-9, indicating that the therapeutic mechanism of EA of back six points for airway remodeling is related to the down-regulation of TGF-8, GM-CSF and IGF-I.更多还原