【作者】 王平；

【导师】 陶功定；

【作者基本信息】 湖北中医药大学 ， 中医基础理论， 2014， 博士

【摘要】 目的：探讨外寒环境因素对正常及肺气虚大鼠一般情况、肺组织结构学、肺组织细胞蛋白差异表达的影响；探究寒邪致病的机理所在。方法：1.选取40只SD大鼠随机分为正常组、寒邪组、肺气虚组、肺气虚加寒邪组，每组10只。肺气虚组模型复制采用气管注射脂多糖与烟熏的复合方法。造模28天后，肺气虚加寒邪组与寒邪组给予寒环境一周，于造模前、后观察记录各组大鼠精神活动、毛色、进食量、饮水量、体重、皮肤、关节及大便情况，连续观察7天。2.寒环境造模完成后各组大鼠于麻醉前12小时禁食、禁水，称量体重。麻醉后开胸取出左肺组织2±0.1g/只，放入10%甲醛溶液中固定，梯度脱水，石蜡切片，常规HE染色，光镜观察结果。3.寒环境造模完成后各组大鼠于麻醉前12小时禁食、禁水，称量体重。麻醉后开胸取出左肺组织10mg/只,每组取3只，进行双向电泳实验、双向凝胶电泳图象分析，肽质量指纹图谱与电喷雾串联质谱鉴定蛋白质。结果：1.与正常组比较，肺气虚组大鼠进食量、饮水量均明显减少，体重增长明显减慢（P<0.05或P<0.01）。与寒邪组、肺气虚组比较，肺气虚加寒邪组大鼠进食量、饮水量明显减少，且体重都较给予外环境前减轻（P<0.05）。肺气虚组大鼠在精神状态、活动、毛色、皮肤、关节及大便等方面均有一定影响，而肺气虚加寒邪组表现尤为明显。2.肺气虚组、寒邪组大鼠病理组织切片则显示肺部出现明显的损伤，肺泡内可见明显的水液潴留，毛细血管充血，炎症细胞浸润。而寒邪加肺气虚大鼠则见间质少量炎细胞侵润，肺泡腔内泡沫细胞聚集，并有肺脓肿形成。3.正常组、寒邪组、肺气虚组与肺气虚加寒邪组每组选三个样本进行差异蛋白组学比较，共计六组，对六对差异蛋白图谱进行蛋白质组学分析,鉴定出65个蛋白,它们在大鼠肺组织蛋白中20个表达上调、45个表达下调。在四组2-DE凝胶上从表达差异蛋白质点中选取点清晰且表达水平改变明显的蛋白质点作为最终质谱鉴定的对象，利用PMF的方法鉴定了7个与肺气虚证可能相关的差异表达的蛋白质、10个与寒邪犯肺可能相关的差异表达的蛋白质。其中转胶蛋白、真核翻译起始因子与肺气虚证有相关性，而热休克蛋白70、血清蛋白前体、纤维蛋白原、苹果酸脱氢酶、柠檬酸合酶则和“内外合邪”大鼠（寒邪作用于肺气虚机体）可能有相关性。结论:1.寒邪对正常组及肺气虚组大鼠的一般情况均有影响，对肺气虚模型大鼠影响更为明显。2.对正常组及肺气虚大鼠肺组织有不同的损害，且寒邪在一定程度上能明显加重肺气虚大鼠肺组织损害的程度。3.初步证实内外因素作用下差异蛋白质交集的存在；并且此种差异蛋白质的功能主要涉及机体激素调节、免疫应答、信号传导、细胞骨架、物质代谢等方面。该结果为研究寒邪伤肺的蛋白质学致病机制提供了科学研究基础资料，亦为今后进一步研究疾病的发生发展奠定了的蛋白组学理论基础，将来也有可能为临床上疾病特别是外感疾病的诊断提供分子生物学方面的诊断标准，具有理论创新和临床指导意义。更多还原

【Abstract】 Objective：To observe effects of cold enviroment on the normal andthe general condition of the rats lung qi deficiency, lung tissuepathology, differnce of cell protein in lung tissue expression. Tostudy the research on the mechanism of cold diseases.Methods:1.A total of40SD rats were randomly divided into controlgroup, cold pathogen group, lung qi deficiency group,lung qideficiency and cold pathogen group, with10rats in each roup. Lungqi deficiency group model replication using composite method byintratracheal injection of lipopolysaccaride and smoked. Deficienyof lung qi and pathogenic cold and cold evil group were given a coldenviroment for a week after28days modeling, and recoded each ratbefore and after modeling about mental activities, hair color, foodintake, water intake, body weight, skin, joints and stool, whichcontinuous observation for7days.2. All rats in each group beforeanesthesia12hours of fasting, water deprivation,weight after coldmolding completion. Thoracotomy to remove left lung tissue afteranesthesia was2±0.1g from each rat, fixed in10%formic acidsolution gradient dehydration, paraffin section, HE staining andthen use light microscope to observe resuls.3. All rats in eachgroup before anesthesia12hours of fasting, waterdeprivation,weight after cold molding completion. Thoracotomy to remove left lung tissue after anesthesia was10g from each rat,3rats from each group, using two-dimensional electrophorsisanalysis,two-dimensional gel electrophoresis image, peptide massfingerprinting and electrospray tandem by mass spectrometry toprotein identification.Result1.Compared with control group, lung qi deficiency group weresignificantly reduced food intake, water intake, and weight gainsignificantly decreased(P<0.05or P<0.01). Compared with the coldpathogen group and lung qi deficiency group, lung qi deficiency andcold pathogen group in rats were significantly reduced food intake,water intake, and weight was reduced compaired which with externalenvironment (P<0.05). Lung qi deficiency rats were impacted inmental state, activity, fur, skin, jiont and stools, while lung qideficiency and cold pathogen group showed obviously.2.The biopsy of lung qi deficiency group and cold pathogen groupconfirmed pulmonary appear obvopus unjury,with clear waterretention, capillary congestion, inflammatory cell infiltration inalveoli.But cold evil and lung qi deficiency group withinterstitial inflammatory cell infiltration,alveolar accumulationof foam cells and lung abscess.3.Selected3samples from control group, cold pathogen group,lung qi deficiency group and lung qi deficiency with cold pathogengroup, a total of6sets of6differntial protin profiles forproteomics analysis, identified65protein in rat lung tissue,protein20expression, regulation45expression. On four groups of2-DE gel from the protein expression differences point selectionpoints clear and protein expression level change obvious point asthe final mass spectrometry identification object, by using the method of PMF identified seven with pulmonary qi deficiencysyndrome may be related to the differentially expressed proteins,10with cold evil lung may be related to the differentiallyexpressed proteins. Which turn gel protein and eukaryotictranslation initiation factor has correlation with lung qideficiency syndrome, and heat shock protein70, serum precursorprotein, fibrinogen, malic dehydrogenase, and citrate synthase,and "internal and external evil" rats (cold evil effects on lungdeficiency of the body) may have relevance.Conclusion:1.The effects of cold pathogen were obvious in normalgroup and lung qi deficiency group at general conditions, which wasmore obvious in lung qi deficiency group.2.There were different damage to the lung tissue of normal groupand lung qi deficiency group, and cold evil in a certain extent canaggravate lung tissue damage degree in lung qi deficiency group.3.The research preliminary confirmed that internal andexternal factors of differential protein intersection exists,and the function of difference of protein mainly involved inhormone regulation, immune response, signal transduction,cytoskeleton, metabolism etc.The results provide basic data for the study of scientificresearch of pathogenic cold injury lung proteomics pathogenicmechanism, protein for further study of the occurrence anddevelopment of disease laid the theoretical foundation, which mayalso provide molecular biology diagnosis of infection disease forclinical disease especially diagnostic criteria, which weretheoretical innovation and the clinical significance.更多还原