【作者】 韩晓静；

【导师】 陈晓玲；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 特发性肺纤维化（idiopathic pulmonary fibrosis,IPF）是一种原因不明的,以弥漫性肺泡炎、肺泡单位结构紊乱和最终导致肺纤维化为特征的进行性间质性肺疾病。其发病率和死亡率都很高,且确切机制尚不明确。目前IPF治疗主要围绕糖皮质激素、免疫抑制剂/细胞毒药物等,其疗效并不十分满意。因此,加强对肺纤维化发病机制的研究及寻找新的有效药物,具有非常重要的临床意义。罗格列酮（rosiglitazone,RSG）属于噻唑烷二酮类药物,是过氧化物酶体增殖活化受体γ（peroxisome proliferator- activated receptor-gamma,PPAR-γ）的合成配体,临床多用于糖尿病的治疗。近年研究表明PPARγ及其配体除具有降血脂、血糖等代谢调节作用,还具有抑制中性粒细胞等抗炎活性,且能减缓脏器纤维化。作为PPARγ高亲和力配体之一的RSG是否对肺纤维化具有作用,目前尚无报道。为此,本实验采用大鼠气管内注入盐酸博莱霉素（bleomycin,BLM）的方法复制与人肺间质纤维化病理过程相似的动物模型,用RSG进行治疗,观察其疗效,并进一步从RSG对肺纤维化大鼠肥大细胞、致纤维化作用因子TGF-β、CTGF的影响来探讨其抗肺纤维化的作用机理,以期为临床防治肺纤维化提出新的治疗药物并提供实验基础。1. RSG对BLM所致大鼠肺纤维化的影响目的各种致病因素造成的肺组织炎症损伤为肺纤维化的首发因素,炎症诱发肺泡上皮正常重构失败,胶原等细胞外基质（extracellular matrix,ECM）成分堆积过多。文献报道RSG对内毒素所致的急性肺损伤具有保护作用,还可抑制肺成纤维细胞Ⅰ型胶原的分泌,从而我们推测它是否能通过抑制肺炎症及减少胶原的产生对肺纤维化产生影响,本部分实验通过对肺组织形态学染色、胶原含量测定等系列指标来观察RSG对BLM所致大鼠肺纤维化的作用。方法60只SD（♂）健康大鼠随机分成二大组:14天和28天组。14天分为4组:Control组、B+NS14组、B+R14组、R14组,28天分为6组:Control组、B+NS28组、B+R14N14组、B+N14R14组、B+R28组、R28组。每组6只。除Control和R组外各组均向大鼠气管内一次性滴注BLM-A5（5 mg·kg-1）造模,Control和R组气管内注入等体积的生理盐水（normal saline, NS）。造模后第一天分别给各组大鼠灌胃以NS、RSG。RSG溶于NS中,制成混悬液,按3 mg·kg-1·d-1给药;NS为2ml·d-1。B+NS组为模型组,造模后给以NS;B+R组为治疗组,造模后以按不同时间点给以RSG,其中B+R14N14组为前14天给RSG,后14天给NS;B+N14R14组为前14天给NS,后14天给RSG;B+R28组28天全程给药;R组气管内注入NS后给予RSG。Control为正常对照组,气管内注入NS后以NS每日灌胃。各组分别于造模后第14、28天取材,常规固定包埋左肺用于形态学染色,冰冻保存右肺用于羟脯氨酸含量测定。所有数据用均数±标准差（x±s）表示,采用SPSS-13.0统计软件,采用单因素方差分析,多个样本均数间的两两比较用最小显著差法,以P<0.05作为判断差异显著性的标准。结果RSG治疗各组动物一般生存状态及肺组织肉眼外观形态观察均好于模型组。肺组织病理学观察显示,模型组14天和28天大鼠肺组织炎细胞浸润、间质成纤维细胞增生明显,肺泡炎和肺纤维化损伤程度分级明显高于对照组（P<0.01）,RSG治疗各组较模型组肺损伤程度明显减轻（P<0.01）。Masson染色显示模型组被染为亮绿色的胶原纤维较对照组显著增多（P<0.01）,并局部形成绿色的纤维团块,RSG治疗各组胶原染色面积较模型组显著减少（P < 0.01）。R14组与Control组比较无差别。羟脯氨酸含量（胶原含量）测定显示:模型组肺组织羟脯氨酸含量与对照组比较显著升高（P<0.01）,B+R14N14及B+N14R14组都较模型组降低,相比有显著意义（P< 0.01）。R28组羟脯氨酸含量较对照组无差别。由以上结果可以得出,气管滴注BLM能成功诱发大鼠肺纤维化模型,RSG(3 mg·kg^-1·d^-1)对肺纤维化不同进展期进行干预,均具有一定的治疗作用。2. RSG对肺纤维化大鼠肥大细胞的影响目的传统观念认为,肥大细胞（mast cells, MCs）参与IgE介导的过敏性反应。近来多有文献报道, MCs及其释放的介质在肺纤维化中发挥重要作用。本部分实验通过观察RSG对肺纤维化大鼠MCs的影响,旨在探讨MCs在肺纤维化中的作用及RSG抗肺纤维化的作用机制。方法动物实验分组及标本采集同第一部分。肥大细胞用甲苯胺蓝特殊染色。采用单盲法,每张切片随机选取10个高倍不重叠视野,准确计数阳性细胞数,取平均值。结果正常大鼠肺组织肥大细胞很少,主要分布于血管周围疏松结缔组织及胸膜附近;模型组14d肺组织MC数量较对照组明显增多（P<0.01）,在实变的肺间质及血管周围分布密集,体积变大,胞浆内充满嗜碱性颗粒,且能观察到细胞脱颗粒现象。至28d肺组织MC数量更多,光镜下见肺间质中尤以纤维化区域MC密集分布,脱颗粒现象活跃。血管旁结缔组织增宽,其中散在MC增多,沿胸膜分布的MC也增多,肺泡腔中偶见MC。RSG治疗各组MC数量较模型组明显减少,仅在血管旁结缔组织中增多,肺间质中MC数量并不多（P<0.05;P<0.01）,R组与Control组比较无差别。以上结果表明,BLM诱导的大鼠肺纤维化肺组织中肥大细胞明显增多,提示肥大细胞与肺纤维化关系密切; RSG通过抑制肥大细胞增生从而减少其释放的介质而减缓纤维化,可能为其抗肺纤维化的作用机制之一。3. RSG对肺纤维化大鼠TGF-β、CTGF的影响目的细胞因子网络失衡是肺纤维化形成发展的重要机制之一。目前已知在参与肺纤维化形成的各种细胞因子中,转化生长因子-β（transforming growth factor-β,TGF-β）是最重要的强效致纤维化作用因子,在ECM生成和沉积调节中起关键作用。其下游作用因子结缔组织生长因子（connective tissue growth factor,CTGF）是近年发现的一种新的可刺激成纤维细胞增殖和分泌胶原的生长因子,其过度表达与某些增生性及纤维化疾病的发生发展关系密切。二者均能刺激成纤维细胞（fibroblast,Fb）向肌成纤维细胞（myofibroblast,MFb）转化。Fb转化为MFb是肺纤维化发展的关键步骤。MFb表达平滑肌肌动蛋白（alpha-smooth muscle actin,α-SMA）,因而α-SMA可作为判断细胞表型改变或MFb的特征性标志。文献报道RSG能阻断TGF-β诱导的人肺肌成纤维细胞分化和过量胶原产生,那么我们推测RSG防治肺纤维化的作用与TGF-β及CTGF有关。前面实验已经证实RSG对肺纤维化中MC具有影响, MC表达TGF-β与CTGF,但在BLM诱导的大鼠肺纤维化中它们之间关系如何尚无报道。顺着这一研究思路,本部分实验采用肺组织连续切片,用免疫组织化学方法及甲苯胺兰染色观察RSG对BLM致肺纤维化大鼠肺组织中TGF-β₁、CTGF、α-SMA、MC的表达情况,以期能更好地研究四者在肺纤维化中的作用及它们之间的相互关系,进一步阐明RSG抗肺纤维化的作用机制。方法动物实验分组及标本采集同第一部分。用免疫组织化学方法观察大鼠肺组织中TGF-β₁、CTGF、α-SMA的表达情况,MC用甲苯胺兰染色。采用单盲法,每张切片随机选取10个高倍不重叠视野,JEDA-801D形态学图像分析系统自动选取染为棕黄色的阳性区域,计算平均光密度值及阳性区域面积百分比。结果TGF-β₁:对照组及R组大鼠肺间质未见明显表达,仅在支气管上皮细胞、血管内皮细胞弱表达。模型组TGF-β₁表达明显增多,主要位于支气管上皮细胞、成纤维细胞、肥大细胞、血管内皮细胞、肺泡巨噬细胞等细胞的胞浆中,与Control组相差非常显著（P<0.01）。B+R治疗各组TGF-β₁表达部位同模型组,但表达明显减少（P<0.05,P<0.01）,R组TGF-β₁表达较Control组无差别。CTGF:对照组及R组大鼠肺组织中CTGF表达弱到无,仅在支气管上皮细胞弱表达。模型组CTGF表达明显增多,部位与TGF-β₁趋于一致,主要位于支气管上皮细胞、成纤维细胞、肥大细胞、Ⅱ型肺泡上皮细胞及肺泡巨噬细胞中,与对照组相差显著（P<0.01）。B+R治疗各组CTGF表达部位同模型组,但表达水平明显减弱（P<0.05,P<0.01）,R组CTGF表达较Control组无差别。α-SMA:对照组及R组α-SMA表达在肺血管内壁及支气管壁平滑肌上,肺实质未见明显表达。模型组除血管壁及支气管壁平滑肌表达α-SMA外,肺泡壁、增厚的肺泡间隔中表达也明显增多,与对照组相差显著（P<0.01）。B+R治疗各组α-SMA表达也增多,但介于对照组及模型组之间,与模型组比较有意义（P<0.05,P<0.01）,R组α-SMA表达与Control组比较无差别。BLM所致肺纤维化大鼠肺组织中TGF-β₁、CTGF、α-SMA表达明显上调,MFb、MC大量表达TGF-β₁、CTGF,提示TGF-β₁、CTGF、MFb、MC四者相互联系、相互影响,与肺纤维化发生发展关系密切。RSG通过抑制肺组织Fb转化为MFb,降低CTGF蛋白的表达,进一步影响TGF-β、的活性,从而减少ECM过度沉积,可能是RSG防治肺纤维化的作用机制之一。结论1.气管滴注BLM能成功诱发大鼠肺纤维化模型,RSG3mg·kg^-1·d^-1对不同进展期肺纤维化进行干预,均具有一定的治疗作用。2. BLM所致肺纤维化大鼠肺组织中肥大细胞明显增加,RSG通过抑制肥大细胞增生从而减少其释放的介质而减缓纤维化,可能为其抗肺纤维化的作用机制之一。3. BLM所致肺纤维化大鼠肺组织中TGF-β₁、CTGF、α-SMA表达明显上调,MFb、MC大量表达TGF-β₁、CTGF,提示TGF-β₁、CTGF、MFb、MC四者相互联系、相互影响,与肺纤维化发生发展关系密切。RSG通过抑制肺组织Fb转化为MFb,降低CTGF蛋白的表达,进一步影响TGF-β的活性,从而减少ECM过度沉积,可能是RSG防治肺纤维化的作用机制之一。更多还原

【Abstract】 Idiopathic pulmonary fibrosis（IPF） is a progressive and interstitial lung disease of unknown etiology that is characteri- zed with diffuse alveolitis and alveolar structure destruction which finally lead to fibrosis. Diseases characterized by IPF cause significant morbidity and mortality.The pathogenesis of IPF is not yet clarified clear now. Sadly,there are few if any effective therapies.The current therapeutic drugs such as glucocorticoids, immunosuppressive agent or cytotoxic drugs are not satisfactory. Therefore the development of successful strategies to prevent pulmonary fibrosis remains an urgent challenge in the clinic.Thiazolidinedione rosiglitazone（RSG）, a potent synthetic agonist of peroxisome proliferator-activated receptor-gamma （PPAR-γ）, is involved in metabolic regulation and insulin resistance therapy. Recent studies have demonstrated that activation of PPARgamma and PPAR-γagonists have anti- inflammatory activities and may have potential as antifibrotic agents. RSG is recognized as a highly selective synthetic ligand for the PPAR-γ,but the effects of RSG on pulmonary fibrosis are not reported until now.In the present study,the model of pulmonary fibrosis（PF） was established by a single intratracheal instillation of bleomycin（BLM） in rats.We observed the effects of RSG on BLM-induced PF. Then we also investigate the possible mechanisms about the effects of RSG on the processes of lung fibrosis in rats by deterning mast cells （MCs） and the expression of profibrotic cytokines transforming growth factor beta （TGF-β） and connective tissue growth factor （CTGF） in lung tissues.This research aims to provide theory foundation for elucidating pathogenic mechanisms of PF and seek novel and more precisely targeted therapies.1 The effect of rosiglitazone on pulmonary fibrosis induced by bleomycin in rats .Aim: The inflammatory response is the initial response following many injury challenges in the progression of lung fibrosis.The response induces an aberrant remodeling of the alveolar epithelial cells and ultimately causes the exaggerated accumulation of extracellular matrix （ECM） components in the interstitial. It has been reported that RSG can reduce acute lung injury in endotoxemic rats. we asked whether RSG is also attenuating lung fibrosis through suppressing lung inflam- mation and reducing collagen deposition. In this study, we extensively investigate the effects of RSG on BLM-induced pulmonary fibrosis in rats determined by observing histomorphology stain and the measurement of hydroxypro- line（HYP） content.Methods: Sixty-two male Sprague-Dawley （SD） rats were randomly divided into two groups: 14-day group and 28-day group. 14-day group was re-divided into 4 subgroups: Control group, B+NS14 group, B+R14 group and R14 group. 28-day group was re-divided into 6 subgroups: Control group, B+NS28 group, B+R14N14 group , B+N14R14 group and R14 group.（6 rats in each group）.The model of pulmonary fibrosis was established by a single intratracheal instillation of BLM-A(55 mg·kg^-1)except Control group and R group,which were injection the same amount of normal saline （NS）.After intratracheal instillation of BLM for 24 h, rats received RSG (3mg·kg^-1)or NS 2ml by gavage per day on different time respectively. B+NS groups were model groups and treated with NS after instillation of BLM. B+R groups were therapy groups and treated with RSG after instillation of BLM.B+R14N14 group was given RSG from 1st day to 14th day,and then given NS from 14th day to 28th day,whereas B+N14R14 group was treated in the opposed way. B+R28 group was treated with RSG for 28 days. R groups were given RSG 14 days or 28 days after instillation of NS. The control group injected and treated with NS.On the 14^th , and 28^th day after intervention,6 rats of each group were killed and their left lungs were collected to undergo light microscopy, right lungs were taken out to measure the content of HYP. Sections were stained with H&E and Masson’s trichrome for collagen identification.Data were entered into a database and analyzed using SPSS software. Group mean values and standard deviations were calculated. After homogeneitic analysis, homogeneous data were analyzed with one-way analysis of variance and a post hoc test of least significant difference （LSD）. The statistical significance level was set at P<0.05.Results: As for living state and lung tissue shapes of rats during the study period, RSG therapy groups were better than the model groups. The histopathologic findings showed: the control group displayed normal structure and no pathologic changes under a light microscope.In the model group on 14th day, severe edema, large amounts of inflammatory cells including neutrophils and lymphocytes in both alveoli and interstitium and fibroblasts proliferation were observed.On 28th day after treatment of BLM, fewer inflammatory changes and foci of collagen or large fibrous areas deposition were observed. Furthermore, marked histopathologic changes, such as, collapsed alveolar spaces were seen.The grades of alveolitis and fibrosis on 14th day and 28th day model groups were upregulated, compared with control group（P<0.01）. RSG therapy groups had shown more obvious attenuation than model group（P<0.01）. Masson stains showed that the area of collagen in model group was increased obviously（P<0.01）and B+R groups displayed less fibrotic lesions compared to it（P <0.01）.R groups were similar to the control group.The content of lung HYP of rats increased more significantly in model group than that in control group（P<0.01）. B+R14N14, B+N14R14 and R28 group decreased obviously versus model group respectively（P<0.01）.These data show that the model of PF was successfully established by a single intratracheal instillation of BLM in rats.RSG (3 mg·kg^-1·d^-1)may have protective effects during the different development of PF induced by BLM.2 The effect of rosiglitazone on MCs in pulmonary fibrosis of rats induced by bleomycin .Aim: Mast cells（MCs） have been traditionally thought to be involved in IgE-associated immediate the allergic response. However, recent studies suggest that MCs play a vital role in pulmonary fibrosis by releasing many cytokines and chemokines.The aim of this study was to investigated the effect of MCs and role of RSG on PF induced by BLM.Methods: Animal groups and lung tissue samples collection in this part were same as that in part 1. MCs were displayed by toluidine blue specific stain.Results: In normal rat lungs there were a few MCs distributed in the loose connective tissue areas close to the vessel and along pleural membrane. In the 14th day model group the number of MCs in the lungs increased significantly （P<0.01）, and they distributed densely in the lung interstitium areas and around the vessels. On 28th day, the body of MC became bigger and basophilic stippling filled the intracytoplasm.We also observed degranulation of mast cells.The number of MCs maintained a higher level and was distributed densely in fibrous areas. B+R groups decreased the number of MCs obviously in lung tissues （P<0.01）, compared with model group.There were no statistical differences in MCs between R group and Control group.These results demonstrate that the number of MCs in the lung fibrotic tissues induced by BLM increased significantly. Our findings confirm the active participation of mast cells in the progression of pulmonary fibrosis in rats. Administration of RSG could inhibite lung fibrotic progression by preventing infiltration and degranulation of mast cells.3. The effect of rosiglitazone on TGF-β₁,CTGF,α-SMA in pulmonary fibrosis of rats induced by bleomycin .Aim: Cytokines disequilibrium is one of important mechanisms involved in pulmonary fibrosis genesis.It is well recognised that TGF-βplays a crucial role in promoting fibrosis among numerous cytokines.It contributing to the ECM pro- duction and deposition. CTGF, a more recently identified potent profibrotic mediator, acts downstream and in concert with TGF-beta to drive fibrogenesis. Significant upregulation of CTGF has been reported in fibrogenic diseases, including IPF, and is partly responsible for associated excessive fibroblast proliferation and extracellular matrix deposition. TGF-βand CTGF may stimulate the differentiation of myofibroblasts（MFb） during fibrosis. MFbs are one of the key effector cells in pulmonary fibrosis and are the primary source of extracellular matrix production. MFb express alpha-smooth muscle actin （α-SMA） de novo which can be as a marker of MFb or MFb differentiation .It is reported that RSG can inhibit TGF-beta induced pulmonary MFb differentiation and collagen production. We hypothesized that the effect of RSG on PF is associated with TGF-βand CTGF. The study in part 2 has been demonstrated that treatment with RSG inhibits lung MCs,so we asked whether MCs can express TGF-βand CTGF and mediate them to promote fibrogenic effects in a murine lung fibrosis model.In following study, serial sections were cut and stained with immunohistochemistry and toluidine blue.We examined the expression of TGF-β₁、CTGF、α-SMA、MC on BLM-induced lung fibrosis to better elucidate their correlation and interaction.Methods: The expression of TGF-β₁、CTGF、α-SMA and MCs in lung tissues was observed by immunohisto- chemistry and toluidine blue stain. The expression of TGF-β1、CTGF、α-SMA will be analyzed by JEDA-801D image analytical system.Results: TGF-β₁:In Control group and R group,TGF-β₁protein expressed weakly in bronchial epithelial cells and vascular endothelial cell（VEC） in lung tissues. In model group, TGF-β₁ showed a high level expression which distributed in the endochylema of bronchial epithelial cells,Fb,MCs, alveolar macrophage（AM） andVEC（P<0.01）,whereas that in B+R groups decreased significantly（P<0.01）. R group was similar to control group. CTGF: In the normal lung, expression of CTGF was sparsely distributed. But in model group CTGF expressed highly which localized in bronchial epithelial cells, Fb, MCs, typeⅡalveolar epithelial cells, AM and VEC（P<0.01）. The B+R therapy groups were similar to model group on the expression place, but expression level decreased significantly compared with i（tP<0.05,P<0.01）. R group no difference than control group.α-SMA: In Control group and R group,α-SMA expressed in smooth muscle of bronchial wall and vessel wall whereas lung parenchyma had nowhere to express.In model groupα- SMA expressed highly in alveolar wall and incrassate alveolar septum and increased obviously in comparison with that in control group（P<0.01）. B+RSG therapy groups decreased compared with model group（P<0.05 or P<0.01）. R group no difference than control group.These data show that MCs express TGF-β₁ and CTGF in BLM-induced rats,suggesting TGF-β₁, CTGF, MFb and MCs may be important mediators in lung fibrogenesis. RSG downregulate the expression of TGF-β₁ and CTGF protein and reduce myofibroblast differentiation in rat lungs induced by BLM.Thus the production of exaggerated accumulation of ECM components is lessened. These findings may be one of the possible mechanisms of RSG attenuat pulmonary fibrosis in different development period .Conclusion 1. The model of pulmonary fibrosis was successfully established by a single intratracheal instillation of BLM in rats.RSG (3 mg·kg^-1·d^-1)may have protective effects during the different development of pulmonary fibrosis induced by BLM.2. The number of MCs in the lung fibrotic tissues induced by BLM increased significantly.Our finding confirm the active participation of MCs in the progression of pulmonary fibrosis in rats. Administration of RSG could inhibite lung fibrotic progression by preventing infiltration and degranulation of mast cells.3. The study shows that MCs express TGF-β₁ and CTGF in BLM-induced rats,suggesting TGF-β₁, CTGF,MFb and MC may be important mediators in lung fibrogenesis.RSG downregulates the expression of TGF-β₁, CTGF protein and reduce myofibroblast differentiation in rat lungs induced by BLM.Thus the production of exaggerated accumulation of ECM components is lessened. These findings may be the possible mechanisms of RSG prevention and cure PF in different development period .更多还原