【作者】 邹万成；

【导师】 张六通； 邱幸凡；

【作者基本信息】 湖北中医学院 ， 中医基础理论， 2009， 博士

【摘要】 目的：研究ECM沉积在IPF纤维化期与疾病进行性发展的关系、IPF纤维化期的病因病机和治法方药、益气通络解毒汤延缓或阻断IPF纤维化期进行性发展的机制，进而探讨ECM与络脉络病的相关性。方法：（1）理论探讨：通过撷菁摘要、去粗存精的文献整理，运用求同存异的对比研究方法，在总结络脉络病的中医学内涵和现代医学阐释、ECM沉积在IPF进行性发展中的作用的基础上，从理论上探讨ECM与络脉络病的相关性、IPF纤维化期的病机，并为从ECM的角度寻求中医药防治肺纤维化的理论依据。（2）实验研究：①建立小鼠肺纤维化模型；②以呼吸频率、体重等观测IPF模型小鼠行为学变化；③病理形态学定量分析小鼠病理学改变；④免疫组化检测coll-1、FN、整合素α5β1、TGF-β1、bcl-2、bax在肺组织中的分布、积分光密度法计算coll-1、FN、整合素α5β1、TGF-β1、bcl-2、bax在肺间质中的阳性面积的IOD值，探寻coll-1、FN与整合素α5β1、TGF-β1、bcl-2、bax在肺间质中的相互关系。结果：（1）行为学：中医治疗组较其它经造模各组在呼吸频率、体重增长、肺系数方面有明显改善，P＜0．05。（2）病理形态学：模型1组有轻到中度的肺泡炎，肺泡腔及肺间质内有一定数量的炎性细胞浸润，毛细血管充血，有点片状出血灶，间质大量成纤维细胞增生、胶原纤维沉积，呈中至重度肺纤维化改变，部分肺泡结构紊乱。模型2组炎症较模型1组减缓，P＜0．05，纤维化加重，P＜0．05。中医治疗组炎症最轻，分别与其它经造模各组比较，P＜0．05；纤维化与模型1组比较无明显差异，P＞0．0 5，与模型2组比较，有明显改善，P＜0．05。西药治疗组在炎症、纤维程度、灰度分区方面与模型2组均无区别。（3）coll-1、FN免疫组化结果：经造模的各组小鼠肺间质内可见coll-Ⅰ呈局灶性、斑片状、条索状分布，较假手术组明显增多，均为P＜0．01；模型2组比模型1组沉积更多，P＜0．05；中医治疗组与模型1组比较无明显变化，P＞0．0 5；西药治疗组与模型2组比较，无明显改善，P＞0．0 5；中医治疗组较模型2组明显改善，P＜0．05。假手术组小鼠肺组织中FN主要分布于血管、支气管周围、肺泡间隔以及肺泡囊开口处，呈细纤维状。中医治疗组与假手术组相似，模型1组、模型2组、西药治疗组在间质细胞呈阳性表达，沉积的ECM中FN亦呈阳性着色，呈不规则点、线状，分布较稀疏，模型1组、模型2组、西药治疗组两两比较，无显著差异，为P＞0．05；三者分别与假手术组、中医治疗组比较，均为P＜0．01。（4）肺间质中整合素α₅β₁免疫组化定量检测：假手术组与中医治疗组比较，P＜0．05，有显著差异；与其它各组比较差异更显著，均为P＜0．01。模型2组较模型1组减轻，P＜0．05；中医治疗组较模型1组、模型2组、西药治疗组均轻，P＜0．05。（5）肺间质中TGFβ1表达的免疫组化定量检测：假手术组与中医治疗组比较，P＜0．05，有显著差异；与其它各组比较，均为P＜0．01；模型1组较模型2组减弱，P＜0．05；中医治疗组较模型1组、模型2组、西药治疗组减弱，P＜0．05。（6）各组小鼠肺间质中bcl-2积分光密度：假手术组与中医治疗组有差别，P＜0．05，与其余各组比较均为P＜0．01。模型2组较模型1组增强，P＜0．05，与西药治疗组相似，P＞0．05。中医治疗组较模型1组、模型2组、西药治疗组均减弱，均为P＜0．01。各组小鼠肺间质中bax积分光密度：中医治疗组分别与模型1组、模型2组、西药治疗组比较，均为P＜0．05。（7）肺间质中coll-1、FN与整合素α5β1、TGF-β1、bcl-2、bax的相互关系：coll-1、FN分别与α5β1、TGF-β1、bcl-2在模型1组、模型2组、西药治疗组组内均呈正线性关系，与bax呈负线性关系。在中医治疗组组内coll-1、FN与整合素α5β1、TGF-β1、bcl-2、bax无线性关系。结论：通过系统理论探讨和实验研究表明：（1）ECM作为细胞生存的微环境，其变化并非仅仅是被动的、各种刺激因素作用的结果，它通过整合素的介导也能影响细胞的功能，在IPF纤维化期疾病仍呈进行性发展中扮演了一个重要的角色。其引起的病理变化与络病高度一致，是络病的现代生物学基础之一。（2）益气通络解毒汤能有效抑制博莱霉素诱导的小鼠特发性肺纤维化纤维化期进行性发展，其机理可能是阻断了沉积的ECM与整合素之间的相互促进关系。其治疗结果与恢复络脉之“运行和渗灌气血、运毒排毒、沟通上下内外功能”高度一致，故阻断沉积的ECM与整合素的相互促进作用是益气通络通络法的现代生物学内涵之一。更多还原

【Abstract】 Research the Relevantof Ruomai and RuobingwithExtracellular Matrix from IPFSpeciality:Basic Theory of TCMAuthor:Zou WanchengTutor:Zhang Liutong;Qiu XingfuanObjective:Study the relevance of ECM deposition with progressivedevelopment of the disease,the mechanismsof Yiqitongluo Decoctiondelaying or blockingprogressive development in IPF fibrosisphase,and discuss the relevance of ECM with Luomai and Luobing.Methods:(1) Theoretic discussion:Collecting essence abstract,eliminateing the false and retaining the true,using of thecomparative research methods of seeking common ground whilereserving differences,on the basis of summing up the traditionalchinese medicine connotation of Luomai and Luobing,the role of ECMdeposition in IPF fibrosis phase,to explore the relevance of ECMwith Luomai and Luobing,pathogenesis mechanisms of traditionalchinese medicine in IPF fibrosis phase,and to seek the theoreticalfoundation of traditional Chinese medicine to combat pulmonaryfibrosis.(2) Experimental Study:①Toestablishamousemodel ofpulmonary fibrosis;②To observate behavior change of mouse modelof pulmonary fibrosis from respiratory rate,bodyweight,etal;③Quantitative analysis of pathological histopathological changes inmouse.④Using immunohistochemical method to detect coll-1,FN,integrinα5β1,TGF-β1,bc1-2,bax in the lung tissuedistribution,using integral optical density method to detectcoll-1,FN,integrinα5β1,TGF-β1,bcl-2,bax in the pulmonary interstitial spaceof the positive IOD values,toexplore the mutualrelations of thecoll-1 andFNwith integrinα5β1,TGF-β1,bcl-2,bax in lung interstitium.Results:(1) behavior study:Chinese medicine treatment group hasa marked improvement comparing groups modeled at the respiratoryrate,weight gain,lung coefficient,P＜0.05.(2)Pathomorphology:Model 1 has mild to moderate lung alveolitis,there are a numberof inflammatory cell infiltration,capillary congestion in lungalveolar space and interstitial lung,Punctate even flakyhemorrhage,a large number of interstitial fibroblasts and collagenfiber deposition,was in moderate to severe pulmonaryfibrosis,with structure disorder in part of the alveoles.inflammation in model 2 Group is sligher than the model 1 Group,P＜0.05,but severe in fibrosis,P＜0.05.Inflammation is thelightest in traditional chinese medicine treatment group,respectively compare with othe groups modeled,P＜0.05;fibrosiscompared with the model 1 was no significant difference,P＞0.05,compared with model 2,there is amarked improvement,P＜0.05.Thereare not difference between Western medicine treatment group andmodel 2 group in the inflammation,the degree of fiber,graydistrict.(3)Immunohistochemistry detectionresults of OID value ofColl-1,FN in lung interstitium:The groups modeled can be seeninterstitial Coll-I showed focal,patchy,cord-like distribution,was significantly increased comparing with sham-operated group,P＜0.01,sedimentary in model 2 is more severe than in model 1 Group,P＜0.05;There are no significant change between Chinese medicinetreatment group and model 1 group,P＞0.05;Western medicinetreatment group compared with the model 2 group,There are no significant improvement,P＞0.05;Chinese medicine treatment groupwas improved compared with the model 2,P＜0.05.FN in lung tissuesof Sham-operated group mice is mainly distributed in the bloodvessels around the bronchi,alveoli and alveolar capsule openinginterval,were tenuous fibrosis-like.FN in chinese medicinetreatment group were similar to in sham -operated group,there arepositive expression at stromal cells in model 1 group,model 2 group,western medicine group,there are also positive staining of FN inECMdeposition,showing irregular,linar,more sparse distribution,comparing among the there gropes,was no significant difference,P＞0.05;separately comparing the three groups with sham-operatedgroup,traditional Chinese medicine group,There are significantdiffrence,P＜0.01.(4)Immunohistochemistry detection results ofOID value of integrinα5β1 in lung interstitium:sham-operatedgroup compared with the traditional Chinese medicine treatmentgroup,P＜0.05,there are significant difference;with othergroups,P＜0.01.Model 2 group is slighter than model 1 group,P＜0.05;Chinese medicine treatment group is slighter than model 1 group,model 2 group and western medicine group,P＜0.05.(5)Imunohistochemistry detection results of OID value of integrinα5β1 in lung interstitium:sham-operated group compared with thetraditional Chinese medicine treatment group,P＜0.05,there issignificant difference;with other groups,P＜0.01;Model 2 Groupshow decrease contrast model 2 Group,P＜0.05;Chinese medicinetreatment group show decrease contrast model 1 group,model 2 group,and western medicine group,P＜0.05.(6) Imunohistochemistrydetection results of OID value of bc1-2 in lung interstitium:sham-operated group compared with TCM treatment group show difference,P＜0.05,with the rest of groups,P＜0.01.Model 2 groupwas enhanced contrasting model 1 group,P＜0.05,similar to thewestern medicine treatment group,P＞0.05.Chinese medicinetreatment group were reduced contrasting model 1 group,model 2group,western medicine treatment group,P＜0.01.Imunohistochemistry detection results of OID value of bcl-2 in lunginterstitium:traditional Chinese medicine treatment grouprespectively compared with the model 1 group,model 2 group,westernmedicine group show difference,P＜0.05.(7)the relationship ofcoll-1,FN with integrinα5β1,TGF-β1,bcl-2,bax in lunginterstitium:there are respectively positivelinear relationshipsbetween co11-1,FN andα5β1,TGF-β1,bcl-2,there are negativelinear relationships between co11-1,FNwithbax inmodel 1 group,model 2 group,western medicine,there are no linear relationshipsbetween co11-1,FN withα5β1,TGF-β1,bcl-2,bax in TCMtreatment group.Conclusion:The Theoretic discussion and experimental results showthat:(1)asa themicro-environmentofcellsurvival,ECM’ schangesare not merely passive results inavarietyof stimuli,through theintegrin-mediated it canalso affect the function of cells,playsan important role in progressive development of IPF fibrotic period.The pathological changes caused by it are hightly Consistent withLuobing,it is one of the Basic material of modern biology of Luobing.(2) Yiqitongluo Decoction can effectively inhibt progressivedevelopment of bleomycin-induced pulmonary fibrosis of mouse,itsmechanism is probably blocking the mutal promoting relation betweenECM deposited and integrins.The results of the treatment isconsistent with“recovering Luomai’ s functions of the”running and infiltrating Qi and blood,running and Excreting poison,linking from top to bottom and inside to outside”,so blockingthe mutal promoting relation between ECM deposited and integrinsmaybe is one connotation of the modern biology of Tongluo law.Subject words pulmonary fibrosis;progressivedevelopment;ECM;Luomai and Luobing;Yiqitongluojiedu更多还原