【作者】 向希雄；

【导师】 赵映前；

【作者基本信息】 湖北中医学院 ， 中医临床基础， 2009， 博士

【摘要】 目的畅肺防喘方是课题组多年来治疗哮喘的经验方,应用于临床疗效显著,该方能有效缓解患者支气管痉挛,提高患者生活质量。其免疫作用已有初步探索。本课题拟在前期临床观察和实验研究的基础上,进一步从分子生物学和免疫学水平,研究畅肺防喘方防治哮喘的作用机制。本课题通过体外实验,运用血清药理学方法,研究该方对支气管平滑肌细胞的抑制增殖作用;并通过体内实验,观察该方对哮喘模型大鼠的平喘作用和对其支气管、肺组织形态学的影响,研究其血液、肺泡灌洗液中EOS(嗜酸性粒细胞)、IgE及相关趋化因子Eotaxin(嗜酸性粒细胞趋化蛋白)、ICAM-1(细胞间粘附分子-1)表达水平的变化。进一步从细胞和分子生物学水平探讨畅肺防喘方治疗哮喘的作用机理,从而为开发临床防治哮喘的有效中药制剂奠定基础。方法1、体内实验方法:取大鼠支气管肺泡灌洗液(BALF)进行细胞计数和分类,对其上清液以双抗夹心酶联免疫吸附(ELISA)法进行细胞因子Eotaxin、ICAM-1含量的测定;血细胞计数分类,放免法检测血中IgE含量;制备大鼠支气管及肺组织切片,HE染色,观察气道重构情况;免疫组化法检测肺及支气管局部组织Eotaxin、ICAM-1的表达。2、体外实验方法:含药血清制备;MTT酶标法、免疫细胞化学(ICC)法测支气管平滑肌细胞的增殖水平,以了解该方对抗哮喘气道重构的作用。结果1、体内试验:1.1实验结果显示畅肺防喘方可显著降低WBC数量,尤其是嗜酸性细胞(P<0.01),说明该方可明显改善呼吸道炎性症状。实验结果还表明血清IgE的含量降低,说明其治疗和预防作用可能是通过此途径来实现的,为畅肺防喘方治疗支气管哮喘的临床应用提供了有力的实验依据。1.2 ELISA法结果显示:与正常组比较模型组支气管肺泡灌洗液中Eotaxin、ICAM-1均显著升高,经治疗后,上述指标均显著降低。说明畅肺防喘方可显著抑制哮喘大鼠Eotaxin、ICAM-1表达。前期研究表明,该方对EOS的抑制作用较为明显。EOS浸润是哮喘炎症的显著病理生理特征,EOS向炎症部位趋化、募集,是哮喘病症中重要的病理过程,其过程机制与Eotaxin、ICAM-1等关系密切。结果提示,对Eotaxin、ICAM-1的抑制作用可能是畅肺防喘方治疗哮喘的作用机制之一。1.3肺组织病理切片HE染色显示,抗原激发后模型组肺组织嗜酸性粒细胞和炎症细胞浸润与对照组相比有明显增多(P<0.01)。2、体外实验:本实验发现畅肺防喘方高、中、小剂量组含药血清对支气管平滑肌增殖细胞核抗原(PCNA)表达的平均光密度明显高于对照组(P<0.05),与地塞米松组无显著性差异(P>0.05),它与MTT法观察结果一致。本次实验证明,与对照组比较,畅肺防喘方高、中剂量含药血清组能明显抑制大鼠支气管平滑肌细胞(ASMC)的增殖,与地塞米松含药血清组比较无显著性差异,而畅肺防喘方小剂量含药血清组则无明显作用,考虑可能与药物浓度有关。且随着作用时间的延长,含药血清对支气管平滑肌细胞的抑制作用有增强趋势。说明畅肺防喘方可能通过抑制组胺(His)促ASMC增殖作用而达到防治哮喘的效果,且存在一定的剂量、时间依赖关系。结论1、体内实验:证实了畅肺防喘方的平喘作用(症状改善,血中IgE、EOS,BALF中EOS含量下降,支气管及肺形态学的变化)和作用机制(抑制趋化因子Eotaxin和粘附分子ICAM-1的表达)。2、体外实验:运用血清药理学方法证实了畅肺防喘方有较好的抗哮喘大鼠气道重构的作用(抑制支气管平滑肌细胞增殖)和抑制平滑肌细胞的PCNA的作用。3、明确了宣肺平喘、清热化痰法对哮喘的防治作用机制。4、为进一步开发具有较好疗效的中药复方制剂,提供了实验依据。更多还原

【Abstract】 objectiveThe Prescription of Patecy Lung and preventing asthma,which is used in clinical medicine is preliminary exploration in immunization for many years,and effect was Significantly. Inhibition of value-added for smooth muscle cell of bronchus were studied though Serum pharmacology method in-vitro method assay; Thought in-vivo experiment,the affect of preventing asthma and lung morphology to the model asthma pig mouse which were medicated by this Prescription were observed.The diversification of EOS、IgE、Eotaxin of correlation factor and expression level changes of adhesion molecule ICAM-1 in blood and the bronchoalveolar lavage fluid.Asthma mechanism of prescription of Patecy Lung and preventing asthma was probed through cell and molecular biology level,and this also gave a foundation for the development clinical of traditional Chinese compound prescription to prevent asthma.Methods1、In vivo experimental:first,cells of big mouse were differential counted,then contents of Eotaxin and ICAM-1 of supernatant fluid of cells were surveyed and evaluated by ELISA.Contents of IgE were detected by dfferential blood count and radioimmunoassay. Prepared tissue slice of Lung and bronchus from big mouse,and stained with Hematoxylin Eosin(HE),then Observed gas channel restructuring situation;Gene expression of Eotaxin and ICAM-1 from Lung and bronchial tube partial organization were investigated by immunohistochemistry.2、In vitro method:Including medicine blood serum was prepared;Proliferation level of Smooth muscle cell from bronchus were tested by MTT enzyme label ling and ICC method.Results1、In vivo experimental:1.1 The results were as follows: respiratory tract inflammation symptoms were improved obviously by prescription of Patecy Lung and preventing asthma,because quantity of WBC especially acidophilic leukocyte were cut down significantly decreased(P<0.01).It can also show that treatment and prevention function was realization by reduced the content of blood serum IgE.Those can give the Powerful experiment basis of Clinical practice against Bronchial tube asthma for prescription of Patecy Lung and preventing asthma.1.2 ELISA demonstration:the earlier period research indicated that the EOS was obviously controlled by prescription of Patecy Lung and preventing asthma, because the contents of Eotaxin and ICAM-1 of bronchia alveolus lavage fluid of control group were reduced obviously compared with that of normal group after Treatment.Remarkable pathology physiology characteristic of asthma inflammation was EOS infiltration.EOS hastened and collected to the inflammation spot were important pathology process in asthma illness,which were closely related to Eotaxin and ICAM-1.These results suggested that inhibitory action of Eotaxin and ICAM-1 possibly was as asthma’s mechanism function of prescription of Patecy Lung and preventing asthma.1.3 HE dyeing demonstrated by Lung organization pathological section:After the antigen stimulated,the inflammatory of eosinophilic granulocyte and inflammatory cell of model group lung organization were increased obviously compared with that of the control group(P<0.01).2、Vitro method:This experiment discovered that the PCNA expression’s average light density of blood serum including medicine was obviously higher than that of control group among three groups:normal control,high and low(P<0.05).It had non-significance difference compared with that of Dexamethasone group,and the same result was discovered by MTT method.The experiment proved:ASMC of big mouse was obviously restrained by blood serum group with medicine of high and normal group,and the result was the same as that of Dexamethasone group,but no obviously effect of low group.Maybe medicine density leaded to the result. The ASMC inhibit function had enhancement tendency along with the response time.Maybe explained that asthma was prevented and controlled through inhibition of His and proliferation of ASMC,and certain dosage and time dependence were included.Conclusions1、Vivo experimental:Suppressing cough function(symptoms improved and the content of IgE、EOS and EOS of BALF in blood were dropped. Bronchial tube and lung morphology) and mechanism function(the Eotaxin of chemotactic factor and ICAM-1 of adhesion molecule were controlled)were Confirmed for prescription of Patecy Lung and preventing asthma.2、Vitro experimental:For prescription of Patecy Lung and preventing asthma,Resistance asthma big mouse gas channel blocking function(Suppressed cell multiplication in the smooth muscle of bronchial tube) and mechanism(Suppressed smooth muscle cell’s PCNA) were discovered.3、Freeing lung and relieving asthma and also removing heat-phlegm were defined to asthma.4、Experiment basis for the traditional Chinese compound prescription’s development which relieving asthma was provided.更多还原