【作者】 徐先伟；

【导师】 孙忠人；

【作者基本信息】 黑龙江中医药大学 ， 针灸推拿学， 2010， 博士

【摘要】 目的:1、揭示定喘穴埋针对哮喘大鼠模型信号转导子和转录激活子(STAT6)蛋白含量,嗜酸性细胞特异性趋化因子(Eotaxin)蛋白含量及其mRNA表达,以及支气管肺泡灌洗液(BALF)和血清中白细胞介素-4(IL-4)、白细胞介素-5(IL-5)含量及嗜酸性粒细胞(Eos)数的影响,探索定喘穴埋针治疗支气管哮喘的作用机制,从而为临床治疗哮喘提供新的治疗方法和理论依据。2、揭示定喘穴埋针对各组大鼠不同时点(60m、90m和120m)肺组织中原癌基因蛋白质(c-fos)表达的影响,以明确定喘穴埋针在支气管哮喘治疗中的神经免疫作用途径及机制,进而丰富定喘穴埋针治疗支气管哮喘的科学内涵。方法:选取健康4-6周龄Wistar大鼠136只,采用卵白蛋白(OVA)激发致敏建立哮喘模型。实验一:大鼠随机分为正常对照组8只,哮喘模型组8只,地塞米松组8只,埋针组8只,穴位注射组8只。实验二:大鼠随机分为正常对照组24只、哮喘模型组24只、埋针组24只、埋针并损毁孤束核(NTS)组24只,每组间又随机分为60m、90m和120m三个时点,每个时点大鼠8只。光镜观察肺组织病理形态学的变化,采用免疫组化法和原位杂交法检测哮喘大鼠模型STAT6、Eotaxin蛋白及其mRNA表达,酶联免疫吸附试验法(ELISA法)检测BALF及血清中IL-4、IL-5含量,瑞氏(Wright)染色法测定BALF中WBC数,Eos数及分类数。采用免疫组化法检测大鼠正常对照组、哮喘模型组、埋针组、埋针并损毁孤束核(NTS)组三个时点(60m、90m、120m)c-fos蛋白的表达。结果:1、造模成功后,经干预治疗,埋针组、地塞米松组和穴位注射组与哮喘模型组比较,大鼠激发后6-10min症状明显减轻,引喘潜伏期明显延长,跌倒率显著降低,经统计学处理,有显著性差异(P<0.01);埋针组与地塞米松组相近,无显著性差异(P>0.05);埋针组与穴位注射组比较有显著性差异,具有统计学意义(P<0.01)。穴位注射组与地塞米松组比较有显著性差异,具有统计学意义(P<0.01)。2、哮喘大鼠模型组STAT6蛋白,Eotaxin蛋白及其mRNA表达明显高于正常对照组,经统计学处理,有显著性差异(P<0.01);埋针组、地塞米松组和穴位注射组STAT6蛋白、Eotaxin蛋白含量低于模型组,其mRNA表达明显弱于模型组,经统计学处理,有显著性差异(P<0.01);埋针组与地塞米松组相近,无显著性差异(P>0.05);埋针组与穴位注射组比较有显著性差异,具有统计学意义(P<0.01)。穴位注射组与地塞米松组比较有显著性差异,具有统计学意义(P<0.01)。3、哮喘模型组BALF及血清中IL-4、IL-5含量明显高于正常对照组,经统计学处理,有显著性差异(P<0.01);埋针组、地塞米松组和穴位注射组BALF及血清IL-4,IL-5含量明显低于哮喘模型组,有显著性差异(P<0.01);埋针组与地塞米松组相近,无显著性差异(P>0.05);埋针组与穴位注射组比较有显著性差异,具有统计学意义(P<0.01)。穴位注射组与地塞米松组比较有显著性差异,具有统计学意义(P<0.01)。4、哮喘模型组BALF中Eos绝对值及Eos%较正常对照组显著升高,经统计学处理,有显著性差异(P<0.01);埋针组、地塞米松组和穴位注射组BALF中Eos绝对值及Eos%明显低于模型组,有显著性差异(P<0.01);埋针组与地塞米松组相近,无显著性差异(P>0.05);埋针组与穴位注射组比较有显著性差异,具有统计学意义(P<0.01)。穴位注射组与地塞米松组比较有显著性差异,具有统计学意义(P<0.01)。5、哮喘模型组大鼠肺脏中c-fos表达明显高于正常对照组,经统计学比较,有显著性差异(P<0.01);埋针组大鼠肺脏中c-fos表达明显弱于模型组,以120分钟组最为明显,经统计学比较,有显著性差异(P<0.01);埋针并损毁孤束核(NTS)组大鼠肺脏中c-fos表达弱于模型组,以120分钟组最为明显,经统计学比较,有显著性差异(P<0.01);埋针组哮喘大鼠模型肺脏中c-fos表达弱于埋针并损毁孤束核(NTS)组,以120分钟组最为明显,经统计学比较,有显著性差异(P<0.01)。结论:1定喘穴埋针可以明显减轻大鼠哮喘症状发作程度。2定喘穴埋针通过降低哮喘大鼠模型STAT6、Eotaxin蛋白含量以及下调STAT6mRNA、EotaxinmRNA在气道上皮细胞的表达,降低哮喘大鼠BALF及血清中IL-4、IL-5含量,降低BALF中Eos数,来干预气道炎症,从而达到治疗支气管哮喘的目的。3定喘穴埋针可能通过干预哮喘炎性信息传入的低位整合中枢孤束核(NTS),从而减弱哮喘大鼠模型肺脏中c-fos蛋白的表达,这可能是定喘穴埋针实现其对神经免疫调节作用机制的途径之一。更多还原

【Abstract】 Purpose:1 In order to reveal the effets of method with needle–embedding therapy in Dingchuan point to the asthma rat model STAT6 protein content、Eotaxin protein content and their mRNA and the contents of IL-4、IL-5 and Eos numbers in the serum and BALF,inquiry into the function mechanisms of with needle-embedding therapy in Dingchuan point to bronchial asthma ,provide new treatment methods and theoretical basis for clinical treatment.2 In order to reveal the influence of method with needle-embedding therapy in Dingchuan point of treating the asthma rat model of c-fos protein content in the lung tissues at different points in time( 60m、90m and 120m),reveal the role of the channels and mechanisms with needle-embedding therapy in Dingchuan point to bronchial asthma , so as to improve the scientific connotation of bronchial asthma in Dingchuan point.Methods:Selecting 136 healthy 4-6 week-old Wistar rats were as targets in this experiment,which were randomly divideded into groups. Sensitized with OVA to stimulate the rats to establish the asthma model. 1 Experiment: 40 Wistar rats were randomly divideded into 5 groups, control group(8)、asthma rat model group (8)、dexamethason group (8)、needle– embedding therapy group(8) and acupoint injection group (8).2 Experiment: 96 Wistar rats were randomly divideded into 4 groups. control group(24)、asthma rat model group (24)、needle–embedding therapy group(24)、spoiling NTS group(24),were randomly divided into 60m, 90m and 120m 3 point in time in each group.Observe the pathological changes in lung tissues with light microscope.Adopting immunohistochemistry method and hybrid diz-ation in situ method to examine the expression of asthma rat models of STAT6、Eotaxin protein and their mRNA; Adopting ELISA (enzyme linked immunosorbentassay) to examine the contents IL-4、IL-5 in BALF and serum of asthma rats;The Wright staining examine the WBC number、classification and Eos number in the BALF. Immunohistochemistry method examine expression of the c-fos in the lung tissues asthma rats.Results:1 After the success of modeling, needle-embedding therapy group, dexamethasone group and acupoint injection group, compared with the asthma model group, significantly reduced the symptoms in rats,latent period of asthma was significantly prolonged, falls rate was significantly reduced. Statistically, there are significant differences in them(P<0.01), needle–embe -dding therapy group and dexamethasone group are similar, there was no significant difference between them(P>0.05). There are significant differences between needle-embedding therapy group and acupoint injection group (P <0.01). There are significant differences between acupoint injection groupand dexamethasone group(P <0.01).2 The expression of STAT6 protein、Eotaxin protein and their mRNA were significantly higher in asthmatic rat model group than the control group (P<0.01).STAT6 protein、Eotaxin protein contents were lower in needle embe -dding therapy group、dexamethasone group and acupoint injection group than the model group (P<0.01), theirs mRNA expression was significantly weaker than the model group, Statistically, there were significant differences in them (P<0.01). Needle-embedding therapy group and dexamethasone group were similar, there was no significant difference between them(P>0.05). There are significant differences between needle-embedding therapy group and acupoint injection group (P <0.01). There are significant differences between acupoint injection groupand dexamethasone group(P <0.01).3 In bronchoalveolar lavage fluid (BALF) and serum levels of IL-4、IL-5 in asthma model group, were significantly higher than the normal control group(P<0.01).The contents of IL-4、IL-5 in BALF and serum were signify -cantly lower than the asthma model group in needle-embedding therapy group、dexamethasone group and acupoint injection group(P<0.01). There are significant differences between needle-embedding therapy group and acupoint injection group (P <0.01). There are significant differences between acupoint injection groupand dexamethasone group(P <0.01).4 The absolute value of Eos in BALF and Eos% was higher in asthmatic model group than the normal control group(P<0.01). The absolute value of BALF in the Eos and Eos% was significantly lower in needle-embedding therapy group、dexamethasone group and acupoint injection group than the model group(P<0.01). Needle-embedding therapy group and dexamethasone group are similar, there is no significant difference between them(P>0.05). There are significant differences between needle-embedding therapy group and acupoint injection group (P <0.01). There are significant differences between acupoint injection groupand dexamethasone group(P <0.01).5 In the lungs of asthma model rats c-fos expression was significantly higher than the normal control group(P<0.01).C-fos expression of the lungs of rats in needle-embedding therapy group was significantly weaker than the model group (P<0.01). The 120 minutes group is most obvious in them.C-fos expression of NTS rats in the lungs is weaker than the model group(P<0.01). The expression of c-fos proteins in needle-embedding therapy group was weaker than the spoiling nucleus (NTS) group(P<0.01). The 120 minutes group is most obvious in them.Conclusions:1 Needle-embedding therapy in Dingchuan point can significantly reduce the level of rats with symptoms of asthma attack.2 By reducing the contents of the STAT6 and Eotaxin protein in the asthma rat models,downing the expression of the STAT6 mRNA and EotaxinmRNA in airway epithelial cells,reducing the levels of BALF and serum IL-4, IL-5 to asthmatic rats, reducing the number of Eos in BALF,interfere with airway inflammation, so as to achieve the purpose of treatment of bronchial asthma.3 Needle-embedding therapy in Dingchuan point may interfere with asthma,through inflammatory information passed by the low integration of the central nucleus of solitary tract, reduced c-fos proteins expression in lung tissiues of rats,this may be the one of the ways to achieve its effects on neural mechanisms of immune regulation.更多还原