【作者】 沈丽萍；

【导师】 李小兵；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2010， 硕士

【摘要】 研究目的：检测肺虚痰阻证模型大鼠及正常大鼠白三烯B4(LTB4)、神经生长因子(NGF)、转化生长因子β1(TGF-β1),粒细胞-巨噬细胞集落刺激因子(GM-CSF)、干细胞因子(SCF)等炎性因子的表达,并观察肺虚痰阻证模型大鼠经补肺化痰中药复方治疗后上述指标的动态变化,从炎症介质的角度探讨肺虚痰阻证的形成机制。研究方法：1.动物分组：雄性SD大鼠,体重180-200g,随机分组,正常组8只,肺虚痰阻证模型组14只,肺虚痰阻证治疗组14只。2.造模方法：肺虚痰阻证模型参照文献(王九林,姜惟,卞慧敏.肺虚痰阻病理模型的研制[J].中国中医基础医学杂志,1996,2(4)：44)的方法并加以改进。早上先将大鼠置于较生长环境温度低5℃的冷风下吹10分钟。后将大鼠置于烟熏柜中,每次将0.2g硫磺粉均匀撒于半根清艾条内,点燃烟熏1小时后取出动物,每天上下午定时烟熏大鼠一次,连续42天。平时与正常对照组大鼠饲养于正常环境中,喂食普通饲料。3.给药方法：造模完成后,治疗组予补肺化痰方治疗,按10ml／kg／d灌胃,1次／天；模型组和正常对照组每天灌胃等量体积生理盐水,连续14天。4.取材、染色及图像分析：大鼠麻醉后腹股沟动脉放血活杀,开胸,结扎左肺支气管,取左肺脏用冰生理盐水清洗后,取左肺中叶,置于10%甲醛中固定,常规石蜡包埋,切片,片厚3um,做HE及免疫组化染色。生理盐水2ml注人右侧气管肺组织内,反复抽吸2次,回收率80%以上。将抽取得肺泡灌洗液(BALF)离心(20℃,2000 r／min离心10 min),收集上清液,置-70℃冰箱冻存待用。5.LTB4、SCF、TGF-β1水平的测定：取肺泡灌洗液,采用酶联免疫法(ELISA)检测。具体方法按照试剂盒说明书进行操作。6.大鼠肺组织GM-CSF及NGF表达的检测：用免疫组织化学方法。具体方法按照试剂盒说明书进行操作。7.统计学处理：采用SPSS 16.0软件进行分析,数据以x±s表示,各组间均数比较采用F检验和t检验,P<0.05为有统计学差异。研究结果：1.动物一般情况正常组大鼠：大鼠活泼好动,反应灵敏,毛发洁白光泽,食欲旺盛,无咳嗽、气促、痰鸣等异常体征。模型组大鼠：大鼠活动迟缓、时有咳嗽、咳痰、气促、毛发枯槁发黄、口鼻分泌物增多,食量及饮水量均较正常组多。治疗组大鼠：大鼠毛发较模型组有光泽,有少许咳嗽、咳痰。2.咳嗽分析用浓氨水刺激诱发咳嗽后,3分钟内正常组(1.30±1.059)的咳嗽次数最低,与模型组(3.00±1.054)及治疗组(2.30±0.949)比较差异均有统计学意义(P<0.05)；模型组(3.00±1.054)与治疗组(2.30±0.949)比较差异无统计学意义(P>0.05)。3.体重实验大鼠正常饲养一周后造模,随着造模的进行,模型组大鼠体重增长较正常组明显减缓。造模前(第一周),正常组(235.5±6.12)和模型组(231.57±9.75)大鼠体重差异无统计学意义(P>0.05)；实验最后一周(第七周),正常组(450.13±60.30)和模型组(399.11±41.29)大鼠体重差异有统计学意义(P<0.05)。4.病理切片正常组大鼠：气管、支气管纤毛柱状上皮细胞排列整齐,纤毛排列规则,未见有明显炎症细胞浸润。模型组大鼠：气管、支气管杯状细胞增生,腺体肥大、增生,粘液分泌旺盛,纤毛稀少,管腔内充满大量的淋巴细胞、肺泡巨噬细胞、中性粒细胞、嗜酸性粒细胞及黏液。治疗组大鼠：气管、支气管杯状细胞增生,腺体肥大、增生均较模型组减轻,纤毛较模型组增多,管腔内有少量肺泡巨噬细胞、中性粒细胞、嗜酸性粒细胞及黏液。5.LTB4.SCF.TGF-β1水平的测定结果：大鼠肺泡灌洗液中LTB4模型组(163.944±83.478)>治疗组(156.326±56.388)>正常组(90.820±46.215)；SCF模型组(0.600±0.244)>治疗组(0.573±0.207)>正常组(0.368±0.116)；TGF-β1模型组(391.714±250.639)>治疗组(380.898±312.992)>正常组(89.428±61.380),以上各指标对照组与模型组,对照组与治疗组比较差异均有统计学意义(P<0.05);模型组与治疗组比较差异无统计学意义(P>0.05)。6.大鼠肺组织GM.CSF及NGF表达的检测结果：大鼠肺组织中炎症介质所占百分比GM.CSF模型组(65.710±10.163)>治疗组(51.820±12.505)>正常组(46.250±7.440)；NGF模型组(75.710±13.986)>治疗组(61.820±11.677)>正常组(61.250±16.421),模型组与正常组、治疗组的阳性细胞所占百分比差异均有统计学意义(P<0.05),染色信号强度模型组(+++)与治疗组(+++)相当；虽然正常组和治疗组的阳性细胞所占百分比差异无统计学意义(P>0.05),但染色信号强度治疗组(+++)>正常组(++)。结论：模型组IJB4、NGF、TGF-β1、GM-CSF、SCF水平均较治疗组和正常组上调,提示以上炎症介质均参与肺虚痰阻证的病变过程,同时也提示肺虚痰阻证的病变过程是一个慢性炎症过程。补肺化痰方可降低炎症介质的表达水平,可通过其抗炎作用对慢性炎症起一定的预防作用。补肺化痰方治疗肺虚痰阻证的作用机制之一可能就是通过下调炎症介质的表达水平实现的。更多还原

【Abstract】 Purpose:Detection of lung deficiency and phlegm obstruction model rats and normal rats of leukotriene B4 (LTB4), nerve growth factor (NGF), transforming growth factorβ1 (TGF-β1), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF) such as the expression of inflammatory factors and to observe the lung deficiency and phlegm obstruction model of rat lungs phlegm after the treatment of Chinese herbal compound the dynamic changes of inflammatory mediators from the perspective of lung deficiency and phlegm with the formation mechanism.Methods:1. Animal groups:male SD rats weighing 180～200g, were randomly assigned to the control group 8, lung deficiency and phlegm syndrome group 14, lung deficiency and phlegm obstruction treated group 14.2. Making touch Methods:lung deficiency and phlegm obstruction model according to the literature (Wang Jiulin, Jiang Wei, Hui-Min Bian. Lung deficiency and phlegm pathological model of development [J]. Traditional Chinese Medicine, 1996,2 (4):44) and improved methods. Rats placed in the morning than the first low growth temperature under 5℃in the cold wind blowing for 10 minutes. Smoked cabinet will be placed in rats, each will be spread evenly 0.2g sulfur powder in half of the root Ai Qing, the fire smoke out animals after 1 hour, every day at a time smoked rats, continuous 42 days. Usually with the normal control group reared in normal environments, feeding normal diet.3. Treatment:modeling is complete, the treatment group Treatment of phlegm to the lungs, according to 10ml/kg/d orally,1 time/day; model group and control group were administered equal volume of normal saline, for 14 days.4. Drawn, staining and image analysis:anesthesia, killed at the groin artery bleeding, thoracotomy, ligation of the left lung bronchus, left lung obtained after washing with normal saline ice, take the middle left lung, placed in 10% formalin fixed, paraffin embedding, slicing, slice thickness 3um, so HE and immunohistochemical staining.2ml saline injection on the right tracheal lung tissue, repeatedly pumping two times, more than 80% recovery rate. The pumping to obtain lavage fluid(BALF) centrifugation(20℃,2000r/min centrifugation 10 min), supernatant collected, frozen standby home-70℃refrigerator.5. LTB4, SCF, TGFβ1 in serum:from lavage fluid by enzyme linked immunosorbent assay (ELISA) test. Specific methods to operate according to kit instructions.6. Lung tissue expression of GM-CSF and NGF detection:Using immunohistochemical method. Specific methods to operate according to kit instructions.7. Statistical analysis:SPSS 16.0 software used to analyze the data to x±s, that the number of each group were compared by F test and t test, P<0.05 as significantly different.Results:1. Animals generallyNormal rats:rats lively, responsive, Maofa Jie Bai Guangze, appetite, no cough, shortness of breath, phlegm and other abnormal signs.Model group:rats activity slow, sometimes cough, sputum, shortness of breath, hair, withered and yellow, mouth and nose secretions increase, food intake and water intake more than normal group.Treated group:Rats in model group than shiny hair, a little cough, sputum.2. Cough AnalysisCough evoked with concentrated ammonia after 3 minutes of normal group(1.30±1.059) in cough frequency of the lowest, with the model group(3.00±1.054) and treatment group(2.30±0.949) were statistically significant differences (P<0.05);model group(3.00±1.054) and treatment group(2.3±0.949) difference was not statistically significant (P>0.05).3. WeightNormal feeding rats a week after the modeling, with modeling of conduct, model group compared with normal weight gain was significantly slowed. Before modeling (first week), the normal group (235.5±6.12) and model group (231.57±9.75) body weight difference was not statistically significant(P>0.05); experiment last week (7th week), the normal group (450.13±60.30), and model group(399.11±41.29) body weight was significantly (P<0.05).4. PathologicNormal rats:trachea, bronchi, ciliated columnar epithelial cells arranged in neat rows cilia regularly arranged, and no significant infiltration of inflammatory cells.Model group:tracheal, bronchial goblet cell hyperplasia, glandular hypertrophy, hyperplasia, mucus secretion exuberant, rare cilia, the lumen filled with a large number of alveolar macrophages, neutrophils, eosinophils and mucus.Treatment group:tracheal, bronchial goblet cell hyperplasia, gland hypertrophy, hyperplasia than those of model group, model group, more than cilia in the lumen of a small amount of alveolar macrophages, neutrophils, eosinophils and mucus.5. LTB4, SCF, TGFβ1 levels of the measured results:LTB4 in BALF in rats of model group (163.944±83.478)>treatment group (156.326±56.388)>normal group (90.820±46.215); SCF model group(0.600±0.244)> treatment group(0.573±0.207)> normal group(0.368±0.116); TGFβ1 model group (391.714±250.639)> treatment group (380.898±312.992)> normal (89.428±61.380), the above indexes in control group and model group, control group and treatment group differences were statistically significant (P<0.05); model group and treatment group difference was not statistically significant(P>0.05).6.Rat lung tissue expression of GM-CSF and NGF test results:Inflammatory mediators in the lung tissue of the percentage of GM-CSF model group (65.710±10.163)> treatment group (51.820±12.505)> control group (46.250±7.440); NGF model group (75.710±13.986)> treatment group (61.820±11.677)> normal group (61.250±16.421), the model group and normal group, treatment group, the percentage of positive cells was significantly (P<0.05), staining signal strength model group(+++) and treatment group (+++) very; Although the normal group and treatment group the percentage of positive cells was no significant difference (P>0.05), but the signal intensity of the treatment group(+++)> stained normal (++).Conclusion:Model group LTB4, NGF, TGF-β1, GM-CSF, SCF level than the treatment group and normal group increases, suggesting that these inflammatory mediators are involved in the pathological process of lung deficiency and phlegm obstruction, but also indicate that lung deficiency and phlegm obstruction of the disease process is a chronic inflammatory process. Lungs phlegm may lower the expression of inflammatory mediators, through its anti-inflammatory effect on the prevention of chronic inflammation play a role. Treatment of Deficient Bufei phlegm phlegm obstruction may be one mechanism of action is through down the expression of inflammatory mediators achieved.更多还原