【作者】 刘绍霞；

【导师】 张国俊；

【作者基本信息】 郑州大学 ， 内科学， 2011， 博士

【摘要】 肺纤维化是一组以肺间质弥漫性渗出、浸润和纤维化为主要病变的疾病,发病机制迄今尚不十分清楚,主要涉及到多种炎症细胞、免疫细胞及其分泌的介质和细胞因子,在炎症反应过程中出现免疫调节紊乱、氧自由基损伤、胶原代谢失衡等。早期病变基础是急性肺泡炎,在肺泡炎形成过程中多种炎性细胞、免疫效应细胞活化,分泌大量细胞因子,某些细胞因子如肿瘤坏死因子-α(tunor necrosis factor-alpha,TNF-α)和转化生长因子-β1(transforming growthfactor-beta 1,TGF-β1),它们在组织损伤和修复中发挥了重要的作用。因其病因复杂,临床上缺乏有效的治疗手段,所以预后极差。疾病后期为大量的成纤维细胞的病理性增殖及细胞外基质的进行性聚集,逐步取代正常的肺组织结构,出现限制性通气功能障碍、肺一氧化碳弥散量(diffusing capacity of the lung for carbon monoxide,DLco)降低,最终导致呼吸衰竭而死亡。近年来,肺间质纤维化疾病的发病率和病死率呈逐年递增的趋势,给患者及社会带来了极大的危害。机体大多数组织和细胞都可以产生TGF-β1,如淋巴细胞、巨噬细胞、肥大细胞、粒细胞、血小板、气道上皮细胞和成骨细胞等。TGF-β1是一种多效性细胞因子,其生物学功能包括调控胚胎的发育、调节细胞的增殖和分化、调节免疫应答、减轻炎症反应、参与组织的损伤修复和调节细胞外基质的产生和降解等。已有的研究结果显示,在肺间质纤维化的形成及发展的过程中,TGF-β1水平显著升高,经用糖皮质激素或抗TGF-β1治疗后肺间质纤维化的病理程度明显减轻；敲出TGF-β1基因可明显减轻小鼠博来霉素诱导的肺纤维化。因此,TGF-β1被认为是肺间质纤维化形成和发展过程中的最关键的“开关性”细胞因子。目前,TGF-β1在肺间质纤维化中的作用已被众多实验证实,通过阻断TGF-β1的产生或抑制其生物活性能够有效地抑制肺纤维化的发生发展,提示干扰TGF-β1可能是抗肺纤维化治疗较为理想的手段。为了更进一步证实TGF-β1在大鼠肺间质纤维化模型中对疾病进展的延缓作用,本研究首次采用RNAi技术及动物实验,对TGF-β1在肺间质纤维化中的作用进行了全面、系统的研究,得出如下结论：成功构建针对TGF-β1 siRNA腺病毒表达载体并包装得到腺病毒颗粒,并在细胞水平证实其干扰效果；然后用气管内滴入博莱霉素的方法建立大鼠肺间质纤维化模型；通过鼻腔滴入TGF-β1 siRNA腺病毒干预后,肺间质纤维化程度明显减轻；干预后支气管肺泡灌洗液及外周血叶TGF-β1的表达水平不同。该研究旨在为以TGF-β1为靶点的肺间质纤维化的分子靶向治疗提供新的思路和可行性的临床依据。第一部分沉默TGF-β1基因表达的siRNA腺病毒制备及效果分析方法(1)TGF-β1的siRNA的特异性靶序列和无关对照序列的设计：依据GenBank中TGF-β1的基因(NM 000660)序列,使用Clontech公司的RNAi Designer软件,设计出针对TGF-β1的siRNA的特异性靶序列和无关对照序列。(2)pSIREN-Shuttle-TGF-β1 siRNA的构建：将上述设计的序列在一定条件下退火,利用T4 DNA连接酶将退火产物与pSIREN-Shuttle载体连接,分别得到pSIREN-Shuttle-siTGF和pSIREN-Shuttle-siCon载体。(3)siRNA表达盒的制备:利用PI-SceI/I-CeuI双酶切pSIREN-Shuttle-siTGF和pSIREN-Shuttle-siCon即获得siRNA表达盒(siTGF和siCon).(4)TGF-β1 siRNA重组腺病毒表达载体的构建和鉴定：分别将上述表达盒重组入腺病毒载体pAdeno-X,构建重组腺病毒表达载体pAdeno-X-siTGF和pAdeno-X-siCon.然后利用DNA测序证实插入序列。(5)腺病毒载体的包装：利用Pac I酶切重组腺病毒质粒pAdeno-X-siTGF和pAdeno-X-siCon,用LipofectamineTM 2000将线性化的pAdeno-X-siTGF和pAdeno-X-siCon转染至HEK293细胞中,12～14d后,裂解细胞并收集病毒上清,纯化、测定病毒滴度后保存于-80℃备用。(6)干扰效果的测定：将病毒上清感染肺腺癌A549细胞,于48h收集细胞,利用RT-PCR和Western blot方法检测A549细胞中TGF-β1 mRNA和蛋白的表达。结果(1) TGF-β1及对照发卡样siRNA单链DNA寡核苷酸退火后,电泳可见明显条带,位于100bp以下,接近100bp,与设计完全一致。(2)成功构建了pSIREN-Shuttle-TGF-(31 siRNA重组载体。(3)成功构建了pAdeno-X-TGF-β1 siRNA的表达载体。(4) RT-PCR结果显示,siRNA-1预1组和siRNA干预2组与空白对照组、空载体组、无关siRNA对照组相比TGF-β1mRNA的表达水平明显降低,差异有统计学意义(P<0.05),其中siRNA干预2组的沉默效果优于siRNA干预1组,但差异无统计学意义(P>0.05),提示TGF-β1第2019-2037位的编码序列的沉默效果较好；空白对照组、无关siRNA对照组与空载体组间无明显差异(P>0.05),TGF-β1mRNA的表达水平均较高。(5)Western blot结果显示,空载体组、无关siRNA对照组、siRNA干预1组、siRNA干预2组与空白对照组相比,细胞中TGF-β1蛋白的表达水平均有不同程度的升高,差异有统计学意义(P<0.05),其中,空载体组和无关siRNA对照组升高最明显,此两者比较无明显差别(P>0.05); siRNA干预1组和siRNA干预2组与空载体组和无关siRNA对照组相比,细胞中TGF-β1蛋白的表达水平略有降低,差异有统计学意义(P<0.05),其中,siRNA干预2组细胞中TGF-β1蛋白的表达水平下降较siRNA干预1组下降明显。第二部分TGF-β1 siRNA重组腺病毒干预大鼠肺间质纤维化模型的实验研究方法(1)实验动物及分组：雄性SD大鼠75只(购自河南省实验动物中心),体重220g(220±20g),随机分为4组：空白对照组(15只)、模型对照组(20只)、无关siRNA对照组(20只)和siRNA干预组(20只)。(2)大鼠肺间质纤维化模型的建立：用10%的水合氯醛(40mg/kg)腹腔注射麻醉,无菌条件下做颈部正中切口,经气管软骨环朝向心端插入注射器,模型对照组、无关siRNA对照组、siRNA干预组一次性缓慢注入含博莱霉素0.2-0.3ml的生理盐水溶液,继续注入0.3ml空气,以便使药液充分注入肺内,空白对照组滴入等量的生理盐水。注入后立即旋转大鼠,使药液在肺内均匀分部,切口缝合,常规消毒,待大鼠清醒后正常喂养。(3)TGF-β1 siRNA治疗大鼠肺间质纤维化：24h后,空白对照组、模型对照组分别经鼻滴入100μl/鼠的生理盐水,无关siRNA对照组、siRNA干预组分别滴入等量体积的重组腺病毒pAdeno-X-siCon和pAdeno-X-siTGF。分别于造模后第7、14、28d腹腔注射麻醉后,腹主动脉放血法处死大鼠,进行取材,每次各组随机处死5只,分别进行肺组织病理学形态观察及分子生物学检测。(4)TGF-β1 mRNA和蛋白的表达的检测：利用RT-PCR和I Western blot法检测肺组织·户Ⅰ、Ⅲ型胶原及TGF-β1mRNA和蛋白的表达,并采用免疫组化法检测肺组织TGF-β1蛋白的表达。并采用ELISA法检测肺泡灌洗液和外周血中TGF-β1的表达。(5)超氧化物歧化酶活性的测定：利用ELISA法检测肺泡灌洗液和外周血中超氧化物歧化酶(SOD)的表达水平。结果(1)肺组织病理形态学观察：空白对照组各观察时间点肺组织均结构清晰,未见明显的肺泡间隔纤维化增厚等变化。模型对照组及无关siRNA对照组病变基本相似,各观察时间点均可见肺组织结构破坏,纤维化形成,其中第28d纤维化最为显著,主要表现为肺泡间隔不同程度纤维化增厚并伴有大量炎性细胞浸润。siRNA干预组处理后,各观察时间点大鼠肺组织纤维化程度显著较模型组及无关siRNA组轻,其中第28d纤维化减轻程度最为显著。(2)免疫组化法观察大鼠肺组织中TGF-β1蛋白的表达：模型对照组与无关siRNA对照组在各观察期肺组织中TGF-β1蛋白的表达量均无显著性差异(P>0.05),同时二者都较空白对照组显著升高,差异有统计学意义(P<0.05)；siRNA干预组第7d、14d肺组织中TGF-β1蛋白的表达量与空白对照组比较显著升高,与模型对照组和无关siRNA对照组比较显示降低,差异均有统计学意义(P<0.05)；第28dTGF-β1蛋白的表达量显著低于模型对照组和无关siRNA对照组(P<0.05),但仍高于空白对照组,差异无统计学意义(P>0.05)。(3)肺组织中Ⅰ、Ⅲ型胶原及TGF-β1 mRNA刷蛋白的表达：模型对照组、无关siRNA对照组及siRNA干预组各期Ⅰ、Ⅲ型胶原及TGF-β1 mRNA和蛋白的表达量比空白对照组均有显著升高,差异有统计学意义(P<0.05); siRNA干预组与模型对照组和无关siRNA对照组相比有所下降,差异有统计学意义(P<0.05)；模型对照组与无关siRNA对照组各期相比无明显差别(P>0.05)。(4)肺泡灌洗液和外周血中TGF-β1和SOD的表达水平：肺泡灌洗液中,TGF-β1的表达水平,各观察期模型对照组、无关siRNA对照组及siRNA干预组较空白对照组TGF-β1的含量有不同程度的升高(P<0.05); siRNA干预组与模型对照组、无关siRNA对照组相比有所降低(P<0.05)；模型对照组与无关siRNA对照组相比无明显差别(P>0.05)。在外周血中,各期各组间相比TGF-β1和SOD的含量无明显差别(P>0.05)。结论(1)成功构建出TGF-β1 siRNA腺病毒表达载体,并鉴定了2条对TGF-β1表达具有明显抑制作用的靶序列。(2)TGF-β1 siRNA腺病毒表达载体转入肺腺癌A549细胞中能明显抑制A549细胞中TGF-β1 mRNA和蛋白的表达。(3)TGF-β1 siRNA重组腺病毒载体可抑制博莱霉素所致大鼠肺间质纤维化肺组织中TGF-β1 mRNA、TGF-β1蛋白及Ⅰ、Ⅲ型胶原蛋白的表达。(4)TGF-β1 siRNA重组腺病毒载体可使博莱霉素所致大鼠肺间质纤维化肺泡灌洗液中SOD的含量升高。(5)TGF-β1 siRNA重组腺病毒载体可减轻博莱霉素所致的大鼠肺纤维化,为肺间质纤维化的治疗提供了理论依据。)更多还原

【Abstract】 Pulmonary fibrosis is a class of connective tissue disease, which formed by diffuse pulmonary interstitial inflammation induced by a variety of factors, progressive fibrosis, and excessive deposition of extracellular matrix. Cause of its development is not very clear so far, and it may be mainly related to a variety of inflammatory cells, immune cells, alveolar epithelial cells and fibroblasts and the secretion of mediators and cytokines in the inflammatory response in a complex immune disorder arising in the course of oxygen free radical damage, collagen metabolism imbalance and so on. The normal lung tissue is replaced by collagen and extracellular matrix gradually, slowly progress,the pathological features in the early Period are inflammations in the interstitial, alveolar, pulmonary vascular or peripheral airway, which means diffuse alveolar inflammation, alveolar units of structural disorder; later period, a large number of pathological fibroblasts proliferation and extracellular matrix progressive accumulation, gradually replace the normal lung tissue structure, and ultimately reduce pulmonary interstitial fibrosis, leading to restrictive ventilatory dysfunction, decreased diffusing capacity of the lung for carbon monoxide, ventilation/Q imbalance and hypoxemia, The major clinical symptoms are progressive dyspnea, wheezing, chest tightness, shortness of breath, dry cough, and ultimately develop into diffuse pulmonary fibrosis and honeycomb lung, causing respiratory failure and death. In recent years, the morbidity and mortality of pulmonary fibrosis have showed a trend of increasing year by year, bringing great harm to individuals and society. Nowadays, the pathophysiology is not very clear, lacking effective treatment, So lots of research institutions home and abroad have been dedicated to basic and clinical research. In pulmonary fibrosis.how to control it, how to delay its progression by effect measurements, improve quality of life of patients and prolong the lives of patients are the focus and difficulty of today’s medical professionMost tissues and cells in the body can produce transforming growth factor （transforming growth factor-betal, TGF-β1）, such as lymphocytes, macrophages, mast cells, granulocytes, platelets, airway epithelial cells and bone cells and so on. TGF-β1 is a pleiotropic cytokine, and the biological functions include regulation of embryonic development, regulating cell proliferation and differentiation, regulation of the immune response, reduce inflammation, involved in regulating tissue repair and extracellular matrix production and degradation And so on. Previous studies showed that during the formation and development process of interstitial pulmonary fibrosis, the level of TGF-β1 were significantly increased, and after the use of glucocorticoids or anti-TGF-β1 treatment, the pathological of pulmonary fibrosis was reduced significantly. Therefore, TGF-β1 is considered to be the most critical "switch" of cytokines in the process of formation and development of pulmonary fibrosis.The mechanisms of TGF-β1 leading to pulmonary fibrosis are the followings:（1） Chemotaxis of inflammatory cells and induction of inflammatory factors to promote the formation and progression of inflammation;（2） inducing production of mRNA of procollagen, promoting the formation and deposition of collagen, to making the accumulation of extracellular matrix and degradation reducing;（3） In the injury lung area, promoting fibroblast proliferation and inducing it transformate to myofibroblasts; （4） Inducing other fibrosis factors and leading to fibrosis. Therefore, by blocking the production of TGF-β1 or inhibiting its biological activity can effectively inhibit the development of pulmonary fibrosis, suggests that interference with TGF-β1 may be an ideal anti-fibrosis treatment means.With the rapid development in molecular biology and genetic engineering, the progress of RNA interference （RNA interference, RNAi） technology opens up a new field. In clinical molecular targeted gene therapy of disease. It is through the introduction of double-stranded RNA （double strand, dsRNA）, performing the specific sequence of endogenous small interfering RNA （small interference RNA, siRNA） combining with specific proteins to form RNA induced silencing complex, then identifying and cutting homology mRNA targeting post-transcriptional gene silencing occurs, inhibiting the expression of the protein, It is an important method of treatment in the gene level. More and more research shows that, because of its high efficiency, low toxicity, fast, etc., and effectively silencing gene expression in cells, RNAi technology shows great prospect in targeting treatment of diseases.At present, the effect of TGF-β1 in lung interstitial fibrosis has been confirmed by many experiments, and TGF-β1 gene knockout mice with bleomycin-induced pulmonary fibrosis has been reduced obviously. To further confirm the role of TGF-β1 in the rat model of pulmonary fibrosis, in the present study, we first constructed adenoviral TGF-β1 siRNA expression vector and packaged by adenovirus particles, and confirmed the interference effect; then with intratracheal bleomycin method to establish rat model of pulmonary fibrosis, and by nasal instillation of TGF-β1 siRNA adenovirus for therapeutic intervention, research the effect of downregulation of TGF-β1 in pulmonary interstitial fibrosis, and to explore the possible causes.The aim of the study is to provide new ideas and feasible clinical evidence in molecular targeted therapy of pulmonary fibrosis in which TGF-β1 as a target.Part I preparation for siRNA adenovirus silencing TGF-β1 gene expression and Analysis of resultsMethods（1） Design the siRNA specific target sequence and unrelated control sequence for TGF-β1:According to TGF-β1 gene （NM₀00660） sequence of GenBank, using Clontech’s RNAi Designer software to design the siRNA specific target sequence and the unrelated control sequence for TGF-β1. （2） Construction pSIREN-Shuttle-TGF-β1 siRNA:Annealed the above designed sequence under certain conditions, connection the product and the annealing pSIREN-Shuttle vector using T4 DNA ligase, obtaining pSIREN-Shuttle-siTGF and pSIREN-Shuttle-siCon vector.（3） Preparation for siRNA expression cassette:PI-SceI/I-CeuI double digested pSIREN-Shuttle-siTGF and pSIREN-Shuttle-siCon for siRNA cells （siTGF and siCon）（4） Construction and identification of recombinant siRNA adenovirus expression vector for TGF-β1:The expression cassettes were recombined into adenovirus vector pAdeno-X to obtain pAdeno-X-siTGF and pAdeno-X-siCon. DNA sequencing confirmed the insertion sequence.（5） Packaging of recombinant adenovirus expression vector:PacⅠcut recombinant adenovirus plasmid pAdeno-X-siTGF and pAdeno-X-siCon with enzyme. The linearized pAdeno-X-siTGF and pAdeno-X-siCon were transfected to the HEK293 cells with Lipofectamine2000TM, after 12～14d, collection of viral supernatant, the virus titer was purified and stored at -80℃after the backup.（6） Determination of the interference effect:Infected lung adenocarcinoma A549 cells with the virus supernatant and collected the cells after 48h, using RT-PCR and Western blotting to detect TGF-β1 mRNA and protein expression in A549 cells.Results（1） Annealed TGF-β1 and control single-stranded DNA hairpin siRNA oligonucleotides, electrophoretic bands can be seen clearly, are located in the following 100bp, close to 100bp, consistent with the design.（2） Successfully constructed pSIREN-Shuttle-TGF-β1 siRNA recombinant plasmid.（3） Successfully constructed pAdeno-X-TGF-β1 siRNA expression vector.（4） RT-PCR results showed that the expression level of TGF-β1mRNA is significantly lower in siRNA intervention group 1 and 2 compared with the control group （P<0.05）, silencing effects of group 1 is is better than group 1, but the difference was not significant （P>0.05）; Three control groups were not significantly （P> 0.05）and TGF-β1mRNA expression levels were higher.（5） Western blot results showed that the expression levels of TGF-β1 protein were higher in control group than other groups（P<0.05）, model control group and unrelated siRNA control group increased most significantly, this was no difference between the two （P> 0.05）; The levels of siRNA intervention group 1 and 2 were slightly lower than model control group and unrelated siRNA control group（P<0.05） and the decreased in group 2 is obviously.PartⅡThe emperimental study on interference effect of TGF-β1 siRNA recombinant adenovirus in rat model with pulmonary interstitial fibrosisMethods（1） Laboratory animals and subgroup:75 male Sprague-Dawley （SD） rats,weighting 220g （220±20g）,were randomly divided into four groups:blank control group with 15 rats,model control group,unrelated siRNA control group,and siRNA intervention group with 20 rats in each group.（2） The creation of rat model with pulmonary interstitial fibrosis:The rats were narcotized with peritoneal injection of 10% chloral hydrate （40mg/kg） and performed with neck median incision under sterile conditions,with the syringe inserted through the tracheal cartilaginous ring towards the heart.The rats of the model control group, unrelated siRNA control group and the siRNA intervention group were injected with normal saline solution of 0.2-0.3ml bleomycin,and then injected 0.3ml air, so that the medicine was injected into the lungs sufficiently. The rats of blank control group were injected with the same amount of normal saline solution （NS）.After injection, the rats were rotated at once so that the medicine was well-distributed in the lungs. The incision was stitched after routine disinfection. The rats were conducted with normal feeding after being conscious.（3） The treatment of pulmonary interstitial fibrosis rats with TGF-(31 siRNA:After 24 hours, the rats of blank control group and model control group were treated with nasal drip of 100μl per mouse of normal saline solution, and the rats of unrelated siRNA control group and siRNA intervention group were administrated with a same volume of recombinant adenovirus pAdeno-X-siCon and pAdeno-X-siTGF. The animals were killed with depletion of abdominal aorta afer narcotized via peritoneal injection on day 7,14,28 after molding.Then the material was drawn from them.Five rats were randomly executed each time for separate experiments and the pathological form of lung tissue was observed.（4） The detection of the expression of TGF-β1 mRNA and protein:The expression of collagenⅠandⅢ, TGF-β1 mRNA and protein in lung tissue was detected by RT-PCR and Western-blot methods.The expression of TGF-β1 protein in lung tissue was detected by immunohistochemical method.The expression of TGF-β1 in the alveolar lavage fluid and the peripheral blood was quantitatively determined by ELISA method.（5） The determination of the activity of superoxide dismutase:The expression level of superoxide dismutase （SOD） in the alveolar lavage fluid and the peripheral blood was quantitatively determined by ELISA method.Results（1） Pathomorphology of lung tissue:The structure of lung tissue in control group was clear at each observation time, without any significant thickening changes of alveolar septum fibrosis. The pathological changes in model control group and unrelated siRNA control group were basically similar. The damage of lung tissue structure could be seen at each observation time and fibrosis was formed, especially on the 28th day, fibrosis was most significant, mainly characterized as different degrees of thickening of alveolar septum fibrosis accompanied by inflammatory cell infiltration. After the siRNA intervention group was processed, the pulmonary fibrosis of rats was significantly lighter than the unrelated siRNA group at each observation time, of which the degree of fibrosis on the 28th day reduced the most.（2） Immunohistochemistry detedction of the TGF-β1 protein expression in the lung tissue of the rat:The expression levels of TGF-β1 protein in lung tissue showed no significant difference at each observation stage between the model control group and unrelated siRNA control group （P>0.05）, while both increased markedly higher than the control group with statistical significance （P<0.05）; The expression levels of TGF-β1 protein in lung tissue in the siRNA intervention group was higher than that in the control group on 7th and 14th day, while lower than that in the model control group and unrelated siRNA control group, which had statistical significance （P<0.05）; The expression levels of TGF-β1 protein in lung tissue in the siRNA intervention group was markedly lower than that in the model control group and unrelated siRNA control group （P<0.05）, but higher than that in the control group without statistical significance （P>0.05）.（3） The expression of collagenⅠandⅢ, TGF-β1 mRNA and protein in the lung tissue:The expression of collagenⅠandⅢ, TGF-β1 mRNA and protein in the lung tissue at each stage in the model control group, unrelated siRNA control group and the siRNA intervention group was markedly higher than that in the control group （P<0.05）; The expression of collagenⅠandⅢ, TGF-β1 mRNA and protein in the lung tissue at each stage in the siRNA intervention group was lower than that in the model control group and unrelated siRNA control group （P <0.05）; The expression of collagenⅠandⅢ, TGF-pi mRNA and protein in the lung tissue at each stage in the model control group showed no significant difference with that in the unrelated siRNA control group （P>0.05）.（4） The expression level of TGF-β1 and SOD in bronchoalveolar lavage fluid and peripheral blood:The expression level of TGF-β1 in bronchoalveolar lavage fluid at each observation stage in the model control group, unrelated siRNA control group and the siRNA intervention group was higher than that in the control group in different degrees （P<0.05）; The expression level of TGF-β1 in bronchoalveolar lavage fluid at each observation stage in the siRNA intervention group is lower than that in the model control group and the unrelated siRNA control group （P<0.05）; The expression level of TGF-β1 in bronchoalveolar lavage fluid at each observation stage in the model control group showed no significant difference with that in the unrelated siRNA control group （P>0.05）. The expression level of TGF-β1 and SOD in the peripheral blood at each observation stage showed no significant difference in each group （P>0.05）.Conclusion（1） The TGF-β1 siRNA adenovirus vector was constructed successfully. And two target sequences that could obviously inhibit the expression of TGF-β1 were identified.（2） The TGF-β1 siRNA adenovirus vector could inhibit the expression of TGF-β1 mRNA and protein, collagenⅠandⅢin lung tissue of the rat with pulmonary interstitial fibrosis caused by bleomycin.（3） The TGF-β1 siRNA adenovirus vector could increase the expression of SOD in the alveolus lavage fluid of the rat with pulmonary interstitial fibrosis caused by bleomycin.（4） The TGF-β1 siRNA adenovirus vector could inhibit the pulmonary interstitial fibrosis of rat caused by bleomycin.更多还原