罗格列酮对博莱霉素所致大鼠肺纤维化的影响及其作用机制研究
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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-β_1、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-β_1、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-β_1: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-β_1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-β_1表达部位同模型组,但表达明显减少(P<0.05,P<0.01),R组TGF-β_1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-β_1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-β_1、CTGF、α-SMA表达明显上调,MFb、MC大量表达TGF-β_1、CTGF,提示TGF-β_1、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-β_1、CTGF、α-SMA表达明显上调,MFb、MC大量表达TGF-β_1、CTGF,提示TGF-β_1、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。
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-β_1,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-β_1、CTGF、α-SMA、MC on BLM-induced lung fibrosis to better elucidate their correlation and interaction.
Methods: The expression of TGF-β_1、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-β_1:In Control group and R group,TGF-β_1protein expressed weakly in bronchial epithelial cells and vascular endothelial cell(VEC) in lung tissues. In model group, TGF-β_1 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-β_1 and CTGF in BLM-induced rats,suggesting TGF-β_1, CTGF, MFb and MCs may be important mediators in lung fibrogenesis. RSG downregulate the expression of TGF-β_1 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-β_1 and CTGF in BLM-induced rats,suggesting TGF-β_1, CTGF,MFb and MC may be important mediators in lung fibrogenesis.RSG downregulates the expression of TGF-β_1, 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 .
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-β_1、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-β_1、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-β_1: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-β_1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-β_1表达部位同模型组,但表达明显减少(P<0.05,P<0.01),R组TGF-β_1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-β_1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-β_1、CTGF、α-SMA表达明显上调,MFb、MC大量表达TGF-β_1、CTGF,提示TGF-β_1、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-β_1、CTGF、α-SMA表达明显上调,MFb、MC大量表达TGF-β_1、CTGF,提示TGF-β_1、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。
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-β_1,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-β_1、CTGF、α-SMA、MC on BLM-induced lung fibrosis to better elucidate their correlation and interaction.
Methods: The expression of TGF-β_1、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-β_1:In Control group and R group,TGF-β_1protein expressed weakly in bronchial epithelial cells and vascular endothelial cell(VEC) in lung tissues. In model group, TGF-β_1 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-β_1 and CTGF in BLM-induced rats,suggesting TGF-β_1, CTGF, MFb and MCs may be important mediators in lung fibrogenesis. RSG downregulate the expression of TGF-β_1 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-β_1 and CTGF in BLM-induced rats,suggesting TGF-β_1, CTGF,MFb and MC may be important mediators in lung fibrogenesis.RSG downregulates the expression of TGF-β_1, 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 .
引文
1 Douglas W W, Ryu J H, Schroeder D R. Idiopathic pulmonary fibrosis: impact of oxygen and colchicines, prednisone,or no therapy on survival[J].Am J Respir Crit Care Med, 2000,161:1172
2 Wang N,Verna L,Chen NG,etal.Constitutive activation of peroxisome proliferator-activated receptor-γsuppresses pro-inflammatory adhesion molecules in human vascular endothelial cells. J Biol Chem, 2002,277(37): 34176~34 181
3 Ricote M, Li AC, Willson TM, et al. The Peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature, 1998, 391: 79~82
4 Jiang C, Ting AT and Geed B. PPAR-γ agonists inhibit production of monocyte inflammatory cytokines. Nature, 1998, 391:82~86
5 Kon K, Ikejima K, Hirose M, et al. Pioglitazone prevents early-phase hepaticfibrogenesis caused by carbon tetrach- loride[J].Biochem Biophys Res Commun,2002, 291 (1) :55
6 Iglarz M, Touyz RM, Viel EC, et al.Peroxisome Proliferator-Activated Receptor-α_ and Receptor-γ_ Activators Prevent Cardiac Fibrosis in Mineralocorticoid -Dependent Hypertension[J]. Hypertension, 2003,42(4):737~743
7 Zafiriou S, Stanners SR, Saad S, et al.Pioglitazone inhibits cell growth and reduces matrix production in human kidney fibroblasts[J].J Am Soc Nephrol,2005, 16 (3):638~645
8 陈祥银,严仪昭,曾卫东,等.丹参、川芎嗪及糖皮质激素对肺纤维化保护作用的实验观察[J].中华结核和呼吸杂志,1987,10(3):152~154
9 Carlos R ,Mart ha M , Garcia-Alvarez J ,et al. Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ from growth rate ,apoptosis ,and tissue inhibitor of metalloproteinases expression. Am J Respir Cell Mol Biol ,2001 ,24 :591~598
10 Kazuyoshi K,Masayuki K, Takashige M ,et al . Soluble form of Fas and Fas ligand in BAL fluid from patients with pulmonary fibrosis and bronchiolitis obliterans organizing pneumonia. Chest ,2000 ,118 :451~458
11 Liu D,Zeng BX,Zhang SH,etal.Rosiglitazone,a peroxis- ome proliferator-activated receptor-gamma agonist, reduces acute lung injury in endotoxemic rats[J]. Crit Care Med,2005,33(10):2309~2316
12 Burgess HA, Daugherty LE, et al. PPAR-γagonists inhibit TGF- β induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis[J].Lung Cell Mol Physiol,2005,288: 1146~1153
13 Ashcroft T,Simpson JM,Timbrell VI. Simple method of estimating severity of pulmonary fibrosis on a numerical scale[J].J Clin Pathol,1988,41(4):467~470
14 Snider GL. Celli BR, Goldstein RH, et al. Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin.Lung volumes, volume-pressure relations, carbon monoxide uptake, and arterial blood gas studied.Am Rev Respir Dis,1978,117(2):289~297
15 刘兴汉等.博来霉素 A5 引起豚鼠肺间质纤维化的实验研究.中华结核呼吸系疾病杂志.1986,9 (1): 28~29
16 Szapiel SV, Elson NA, Fulmer JD. Bleomycin induced interstitial pulmonary disease in nude athymic mouse. Am Rer Respir Dis1979,120:893~897
17 Thrall RS,McCormick JK,Jack RM,et al. Bleomycin- induced pulmonary fibrosis in the rat.Am J Pathol,1979,95(1):117~130
18 Genovese T, Cuzzocrea S, Di Paola R, Mazzon E,et al. Effect of rosiglitazone and 15-deoxy-Delta12, 14-prosta- glandin J2 on bleomycin-induced lung injury[J].Eur Respir J,2005,25(2):225~234
19 Ameshima S,Golpon H,Cool CD,et al.Peroxisome Proliferator-Activated Receptor Gamma( PPAR-gamma ) Expression Is Decreased in Pulmonary Hypertension and Affects Endothelial Cell Growth. Circ Res,2003, 92:1162~1169
20 Benayoun L , Letuve S , Druilhe A , et al . Regulation of peroxisome proliferator-activated receptor r expression in human asthmatic airways. Am J Respir Crit Care Med , 2001 ,164 :1487~1494
1 Sasaki M,Kashima M,Ito T, et al. Effects of heparin and related glycosaminoglycan on PDGF-induced lung fibroblast proliferation,chamotactic response and metalloproteinases activity.Mediators Inflamm, 2000, 9: 85~91
2 Sugiyama H, Nonaka T,Kishimoto T,Peroxisome proliferator -activated receptors are expressed in human cultured mast cells: a possible role of these receptors in negative regulation of mast cell activation. Eur J Immunol. 2000,30(12):3363~70
3 Maeyama K, Emi M, Chihara K. Role of PPARgamma in inflammatory response related to mast cells. Nippon Yakurigaku Zasshi. 2003,122(4):325~30
4 Kirshenbaum AS,Kessler SW,Goff JP,etal.Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells[J].J Immunol, 1991, 146(5): 1410~1415
5 man ES. The role of mast cells in inflammatory responses in the lung[J].Crit Rev Immunol , 1993, 13: 36~70.
6 Church MK, Levi-Schaffer F. The human mast cell. J Allergy Clin Immunol. 1997 Feb;99(2):155
7 Egozi EI, Ferreira AM, Burns AL, et al. Mast cells modulate the inflammatory but not the proliferative response in healing wounds. Wound Repair Regen. 2003 ,11(1):46
8 Miller HR, Pemberton AD. Tissue-specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut. Immunology. 2002, 105:375
9 Pallaoro M, Fejzo MS, Shayesteh L, et al. Charac- terization of genes encoding known and novel human mast cell tryptases on chromosome 16p13.3. J Biol Chem. 1999,274(6):3355
10 Pereira PJ, Bergner A, Macedo-Ribeiro S, et al. Human beta-tryptase is a ring-like tetramer with active sites facing a central pore. Nature. 1998, 392(6673): 306
11 Dowdall JF, Winter DC, Baird AW, et al. Biological role and clinical implications of mast cells in surgery. Surgery. 2002,132(1):1
12 Akers IA, Parsons M, Hill MR, et al. Mast cell tryptase stimulates human lung fibroblast proliferation via protease-activated receptor-2. Am J Physiol Lung Cell Mol Physiol. 2000,278(1): L193
13 Abe M, Kurosawa M, Ishikawa O, et al. Effect of mast cell–derived mediators and mast cell–related neutral proteases on human dermal fibroblast proliferation and type I collagen production. J Allergy Clin Immunol. 2000 ,106(1 Pt 2):S78
14 Garbuzenko E, Nagler A, Pickholtz D, et al. Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis. Clin Exp Allergy. 2002 Feb; 32(2): 237
15 Mirza H, Schmidt VA, Derian CK, et al. Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell alpha- or beta-tryptases. Blood. 1997, 90 (10):3914
16 Compton SJ, Cairns JA, Holgate ST, et al. The role of mast cell tryptase in regulating endothelial cell proliferation, cytokine release, and adhesion molecule expression: tryptase induces expression of mRNA for IL-1 beta and IL-8 and stimulates the selective release of IL-8 from human umbilical vein endothelial cells. J Immunol. 1998,161(4):1939
17 Sommerhoff CP. Mast Cell Tryptases and Airway Remodeling. Am J Respir Crit Care Med. 2001 Nov 15;164(10 Pt 2):S52
18 Sime PJ,O Reilly KM.Fibrosis of the lung and other tissues:New concepts in pathogenesis and treatment[J].Clin Immunol,2001,99(3):308
19 Lukacs NW,Hogaboam C,Chensue SW,et al. Type1/type2 cytokine paradigm and progression of pulmonary fibrosis[J].Chess, 2001,120(1Sup pl): 5s
20 Kunkel SL, Chensue SW, Lukacs N,et al. Cytokine phenotypes serve as a paradigms for experimental immune-mediated lung diseases and remodeling[J].Am J Respir Cell Mol Biol,2003,29(3 Suppl): s63
21 Wallace WA,Ramage EA,Lamb D,et al.A type2(Th2-type pattern of response predominates in the pulmonary interstitium of patients with cryptogenic fibrosing alveolitis (CEA).Clin Exp Immunol,1995,101: 436~441
22 Furuie H,Yamasaki H,Suga M,etal.Altered accessory cell function of alverlar macrophages:Apossible mechanism for Induction of Th2 secretory profile in idiopathic pulmonary fibrosis.Eur RespirJ,1997;10:787~794
1 Burgess HA, Daugherty LE, et al. PPAR-γagonists inhibit TGF- β induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis[J].Lung Cell Mol Physiol ,2005,288: 1146~1153
2 Coker R K,Laurent G J.Pulmonary fibrosis:cytokines in the balance[J].Eur Respir J,1998,11:1218
3 Roberts A B, Piek E,Bottinger E P,et al.Is Smad3 a major player in singal transduction pathways leading to fibrogenesis?[J]Chest,2001,120(1 Suppl):43s
4 Gauldie J,Galt T,Bonniaud P,et al.Transfer of the active form of transforming growth factor-beta 1 gene to newborn rat lung induces changes consistent with bronchopulmonary dysplasia[J].Am J Pathol, 2003,163 (6):2575
5 Kasper M,Seidel D,Knels L,et al.Early signs of lung fibrosis after in vitro treatment of rat lung slices with CdCI(2) and TGF-beta(1)[J].Histochem Cell Biol,2004, 121(2):131
6 Oemar BS,Luscher TF.Connective tissue growth factor, friend or foe?[J].Arterioscler Thromb VascBiol, 1997, 17 (8):1483~1489
7 Moussad EE, Rrigstock DR.Connective tissue growth factor; what’s in a name? Mol Genet Metab, 2000, 7; 276~292
8 Williams EJ, Gaca MD, Brigstock DR, et al. Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells. J Hepatol, 2000,32(5):754~61
9 Leask A, Sa S, Holmes A, et al. The control of ccn2 (ctgf) gene expression in normal and scleroderma fibroblasts. Mol. Pathol. 2001, 54:180~183
10 Joseph A, Lasky, Luis A, et al .Connective tissue growth factor mRNA expression is upregulated in bleomycin- induced lung fibrosis[J]. Am J Physiol Lung Cell Mol Physiol ,1998,275(2): 365~371
11 Crean JKG,Finlay D,Murphy M,etal.The role of p42/p44 MAPK and protein kinase B in connective tissue growth factor induced extracellular matrix protein production,cell migration, and actin cytoskeletal rearrangement in human mesangial cells [J].J Biol Chem,2002, 277(46): 44187~ 44194
12 Chen MM, Ian A, Abraham,et al.CTGF expression is induced by TGF-beta in cardiac fibroblasts and cardiac myocytes: a potential role heart fibrosis.J Mot Cdl Cndio1,2000,32:180
13 Hu B , Wu Z , Phan SH. Smad3 mediates transforming growth factor-beta-induced alpha-smooth muscle actin expression. Am J Respir Cell Mol Biol. 2003,29(3 Pt 1):397~404
14 Gu L , Zhu YJ,Yang X, et al. Effect of TGF-beta/Smad signaling pathway on lung myofibroblast differentiation. Acta Pharmacol Sin. 2007,28(3):382~91
15 Folger PA, Zekaria D, Grotendorst G, Masur SK. Transforming growth factor-beta-stimulated connective tissue growth factor expression during corneal myo- fibroblast differentiation. Invest Ophthalmol Vis Sci. 2001 ,42(11):2534~41
16 Huang HC,Yang M, Li JZ, Wang HY. Connective tissue growth factor promotes the proliferation of myofibroblast through Erk-1/2 signaling pathway Zhonghua Yi Xue Za Zhi. 2005 , 25;85(19):1322~6
17 Wu SH, Wu XH, Lu C, et al. Lipoxin A4 inhibits proliferation of human lung fibroblasts induced by connective tissue growth factor. Respir Cell Mol Biol. 2006,34(1):65~72
18 Sanlana A,Saxena B,Nancy A,etal.Increased expression of transforming growth factorβ isoforms(β1、β2、β3 ) in bleomycin-induced pulmonary fibrosis.Am Respir Cell Mol Biol ,1995 ,13 ; 34~44
19 Joseph A. Lasky, Luis A, etal. Connective tissue growth factor mRNA expression is upregulated in bleomycin- induced lung fibrosis. Am J Physiol Lung Cell Mol Physiol. 1998,275: 365~371
20 张雷,卢惠苹,赖国祥.结缔组织生长因子在博莱霉素诱导的小鼠肺纤维化中的作用探讨.细胞与分子免疫学杂志,2005,21;290~292
21 Broekelmann TJ ,Limper AH ,Colby TV,et al. Transforming growth factors β1 is present at sites of extracellrar matrix gene expression in human pulmonary fibrosis.Prnc Natl .Acad Sci USA,1991,88:6642~6646
22 Pan LH,Yamauchi K,Uzuki M,et al.Type Ⅱ alveolar epithelial cells and interstitial fibroblasts express connective tissue growth factor in IPF[J]. Eur Repir J,2001,17(6):1220~1227
23 Hamada H,Vallyathan V,Cool CD,et al.Mast cell basic fibroblast growth factor in silicosis[J].Am J Respir Crit Care Med,2000,161:2026
1 Oemar BS,Luscher TF.Connective tissue growth factor, friend or foe?[J].Arterioscler Thromb Vasc Biol, 1997, 17 (8):1483~1489
2 BrigstockDR.The connective tissue growth factor/ cysteine-rich 61/nephroblastoma overexpressed (CCN) family[J].Endocr Rev 1999,20:189
3 Kubota S,Hattori T,Nakanishi T,etal.Involvement of cisacting repressive element(s) in the 3’-untranslated region of human connective tissue growth factor gene.FEB Lett,1999,450(1-2):84~88
4 Bradham DM,Igarashi A,PotterRL,etal.Connective tissue growth factor:acysteine-rich mitogen secreted by human vascuclar endothelial cells is related to the SRC-induced immediate early gene product CEF-10[J].J Cell Biol,1991, 114(6):1285~1294
5 Murray RJ,McDonald NQ,Blundell TL,etal.Topological similaritie in TGF-beta,PDGF and NGF define a super-family of polypeptide growth factors.Structure 1993, 1:153~159
6 Gupta S,Clarkson MR,Duggan J,etal.Brady Connective tissue growth factor:protential role in glomerulosclerosis and tubulointer-stitial fibrosis.Kidney Inter,2000, 58(5): 1389~1399
7 Grotendorst GR.Connective tissue growth factor:amediator of TGF-beta action on fibroblasts[J].Cytokine Growth Factor Rev, 1997,8:171
8 Chen MM, Ian A, Abraham,et al.CTGF expression is induced by TGF-beta in cardiac fibroblasts and cardiac myocytes: a potential role heart fibrosis.J Mot Cdl Cndio1,2000.32:180
9 Duncan MR, Frazier KS, Abremaon S et al. Camective tissue growth factor mediates .transforming growth factor beta-induced collagen synthesis:Down-regulation CAMP. FASEB J,1999,13; 1774
10 Grotendorst GR, Okochi H, Hayashi N. A novel trans-forming growth factor beta response element controls the expression of the connective tissue growth factor gene.Cell Growth Differ, 1996, 7(4):469~480
11 Leask A, Sa S, Holmes A, Shiwen X, et al. The control of ccn2 (CTGF) gene expression in normal and scleroderma fibroblasts. Mol Pathol, 2001,54(3):180~183
12 Chambers RC, Leoni P, Blanc-Brude OP, et al. Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease- activated receptor-1. J Biol Chem 2000;275:35584~91
13 Abraham DJ, Shiwen X, Black CM, et al. Tumor necrosis factor alpha suppresses the induction of connective tissue growth factor by transforming growth factor-beta in normal and scleroderma fibroblasts. J Biol Chem 2000; 275: 15220 ~5
14 Pendurhti UR, Allen KE, Ezban M, et al. Factor VIIA and thrombin induce the expression of cyr61 and connective tissue growth factor, extracellular matrix signaling proteins that could act as possible downstream mediators in factor VIIA x tissue factor-induced signal transduction. J Biol Chem 2000; 275: 14632~41
15 Riser BL, Denichilo M, Cortes P, et al. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol 2000;11:25~38
16 Murphy M, Godson C, Cannon S, et al. Suppression subtractive hybridization identified high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J Biol Chem 1999;274:5830~4
17 Dammeier J, Beer HD, Brauchle M, et al. Dexamethason is a novel potent inducer of connective tissue growth factor expression. Implications for glucocorticoid therapy.J Biol Chem 1998; 273: 18185~90
18 Frazier K,Williams S,Kothapalli D,et al.Stimulation of fibroblast cell growth, matrix production and granulation tissue formation by connective tissue growth factor.J Invest Dermatol,1996,107: 404~411.
19 Hahn A,Heusinger-Ribeiro J,Lanz T,et al.Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton.J Biol Chem,2000,275(48):37429~35
20 Yokoi H,Mukoyama M,Sugawara A,etal.Role of Connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis.Am J Physiol Renal Physiol,2002,282(5):933~942
21 Luo Z, Li H, Zhang J, etal. Effects of human connective tissue growth factor gene transfection on migration of human umbilical vein endothelial cell. Clin Hemorheol Microcirc.2006;34(1-2):185~92
22 David R,Brigstock.The Connective tissue growth factor/ Cysteine-Rich61/Nephroblastoma overexpressed (CCN) family. Endo Reviewe,1999,20(2):189~206
23 Hishikawa K,Oemar BS,Nakaki T.Static pressure regulates Connective tissue growth factor expression in human mesangial cells.J Biol Chem,2001,276(20):16797~16803
24 Shi-wen X, Stanton LA, Kennedy L,etal. CCN2 is necessary for adhesive responses to transforming growth factor-beta1 in embryonic fibroblasts.J Biol Chem.2006, 281(16):10715~26
25 Chen CC,Chen N,Lau LF.The Angiogenic Factors Cyr61 and Connective Tissue Growth Factor Induce Adhesive Signaling in Primary Human Skin Fibroblasts[J].J Biol Chem,2001,276(13):10443~10452
26 Jedsadayanmata A,Chen CC,Kireeva ML,etal.Activation dependent adhesion of human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is mediated through integrin alpha(IIb)beta(3)[J].J biol Chem,1999, 274 (34) :24321~24327
27 Luo Z, Li H, Zhang J, etal. Effects of human connective tissue growth factor gene transfection on migration of human umbilical vein endothelial cell. Clin Hemorheol Microcirc.2006;34(1-2):185~92
28 Lasky JA, Ortiz LA, Tonthat B, et al. Connective tissue growth factor mRNA expression is upregulated in bleomycin-induced lung fibrosis. Am J Physiol, 1998, 275(2 Pt 1):365~371
29 Ricupero DA, Rishikof DC, Kuang PP, et al. Regulation of connective tissue growth factor expression by prostaglandin E(2). Am J Physiol, 1999,277(6Pt1): 1165 ~1171
30 Ricupero DA, Romero JR, Rishikof DC, et al. Des- Arg(10)- kallidin engagement of the B1 receptor stimulates type I collagen synthesis via stabilization of connective tissue growth factor mRNA[J]. J Biol Chem, 2000, 275(17):12475~12480
31 Xiao R, Liu FY, Luo JY,etal.Effect of small interfering RNA on the expression of connective tissue growth factor and type I and III collagen in skin fibroblasts of patients with systemic sclerosis. Br J Dermatol. 2006, 155 (6): 1145~53
32 Folger PA, Zekaria D, Grotendorst G, Masur SK.Transforming growth factor-beta-stimulated connective tissue growth factor expression during corneal myofibroblast differentiation. Invest Ophthalmol Vis Sci. 2001,42(11):2534~41
33 Gu L,Zhu YJ,Yang X, et al.Effect of TGF-beta/Smad signaling pathway on lung myofibroblast differentiation. Acta Pharmacol Sin. 2007,28(3):382~91
34 Folger PA, Zekaria D, Grotendorst G, Masur SK. Transforming growth factor-beta-stimulated connective tissue growth factor expression during corneal myo- fibroblast differentiation. Invest Ophthalmol Vis Sci. 2001,42(11):2534~41
35 Huang HC,Yang M, Li JZ, Wang HY. Connective tissue growth factor promotes the proliferation of myofibroblast through Erk-1/2 signaling pathway. Zhonghua Yi Xue Za Zhi. 2005,25;85(19):1322~6
36 杨 敏,黄海长,李惊子等.结缔组织生长因子增加 TGFB活 化 成 肌 纤 维 细 胞 的 作 用 . 基 础 医 学 与 临 床 . 2005,25(1)25~29
37 Wu SH, Wu XH, Lu C, et al. Lipoxin A4 inhibits proliferation of human lung fibroblasts induced by connective tissue growth factor. Respir Cell Mol Biol. 2006,34(1):65~72
38 黄山英,宋良文,张勇等.CTGF 表达在大鼠放射性肺纤维 化 中 的 作 用 . 中 华 放 射 医 学 与 防 护 杂 志 .2005, 25(3);213~216
2 Wang N,Verna L,Chen NG,etal.Constitutive activation of peroxisome proliferator-activated receptor-γsuppresses pro-inflammatory adhesion molecules in human vascular endothelial cells. J Biol Chem, 2002,277(37): 34176~34 181
3 Ricote M, Li AC, Willson TM, et al. The Peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature, 1998, 391: 79~82
4 Jiang C, Ting AT and Geed B. PPAR-γ agonists inhibit production of monocyte inflammatory cytokines. Nature, 1998, 391:82~86
5 Kon K, Ikejima K, Hirose M, et al. Pioglitazone prevents early-phase hepaticfibrogenesis caused by carbon tetrach- loride[J].Biochem Biophys Res Commun,2002, 291 (1) :55
6 Iglarz M, Touyz RM, Viel EC, et al.Peroxisome Proliferator-Activated Receptor-α_ and Receptor-γ_ Activators Prevent Cardiac Fibrosis in Mineralocorticoid -Dependent Hypertension[J]. Hypertension, 2003,42(4):737~743
7 Zafiriou S, Stanners SR, Saad S, et al.Pioglitazone inhibits cell growth and reduces matrix production in human kidney fibroblasts[J].J Am Soc Nephrol,2005, 16 (3):638~645
8 陈祥银,严仪昭,曾卫东,等.丹参、川芎嗪及糖皮质激素对肺纤维化保护作用的实验观察[J].中华结核和呼吸杂志,1987,10(3):152~154
9 Carlos R ,Mart ha M , Garcia-Alvarez J ,et al. Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ from growth rate ,apoptosis ,and tissue inhibitor of metalloproteinases expression. Am J Respir Cell Mol Biol ,2001 ,24 :591~598
10 Kazuyoshi K,Masayuki K, Takashige M ,et al . Soluble form of Fas and Fas ligand in BAL fluid from patients with pulmonary fibrosis and bronchiolitis obliterans organizing pneumonia. Chest ,2000 ,118 :451~458
11 Liu D,Zeng BX,Zhang SH,etal.Rosiglitazone,a peroxis- ome proliferator-activated receptor-gamma agonist, reduces acute lung injury in endotoxemic rats[J]. Crit Care Med,2005,33(10):2309~2316
12 Burgess HA, Daugherty LE, et al. PPAR-γagonists inhibit TGF- β induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis[J].Lung Cell Mol Physiol,2005,288: 1146~1153
13 Ashcroft T,Simpson JM,Timbrell VI. Simple method of estimating severity of pulmonary fibrosis on a numerical scale[J].J Clin Pathol,1988,41(4):467~470
14 Snider GL. Celli BR, Goldstein RH, et al. Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin.Lung volumes, volume-pressure relations, carbon monoxide uptake, and arterial blood gas studied.Am Rev Respir Dis,1978,117(2):289~297
15 刘兴汉等.博来霉素 A5 引起豚鼠肺间质纤维化的实验研究.中华结核呼吸系疾病杂志.1986,9 (1): 28~29
16 Szapiel SV, Elson NA, Fulmer JD. Bleomycin induced interstitial pulmonary disease in nude athymic mouse. Am Rer Respir Dis1979,120:893~897
17 Thrall RS,McCormick JK,Jack RM,et al. Bleomycin- induced pulmonary fibrosis in the rat.Am J Pathol,1979,95(1):117~130
18 Genovese T, Cuzzocrea S, Di Paola R, Mazzon E,et al. Effect of rosiglitazone and 15-deoxy-Delta12, 14-prosta- glandin J2 on bleomycin-induced lung injury[J].Eur Respir J,2005,25(2):225~234
19 Ameshima S,Golpon H,Cool CD,et al.Peroxisome Proliferator-Activated Receptor Gamma( PPAR-gamma ) Expression Is Decreased in Pulmonary Hypertension and Affects Endothelial Cell Growth. Circ Res,2003, 92:1162~1169
20 Benayoun L , Letuve S , Druilhe A , et al . Regulation of peroxisome proliferator-activated receptor r expression in human asthmatic airways. Am J Respir Crit Care Med , 2001 ,164 :1487~1494
1 Sasaki M,Kashima M,Ito T, et al. Effects of heparin and related glycosaminoglycan on PDGF-induced lung fibroblast proliferation,chamotactic response and metalloproteinases activity.Mediators Inflamm, 2000, 9: 85~91
2 Sugiyama H, Nonaka T,Kishimoto T,Peroxisome proliferator -activated receptors are expressed in human cultured mast cells: a possible role of these receptors in negative regulation of mast cell activation. Eur J Immunol. 2000,30(12):3363~70
3 Maeyama K, Emi M, Chihara K. Role of PPARgamma in inflammatory response related to mast cells. Nippon Yakurigaku Zasshi. 2003,122(4):325~30
4 Kirshenbaum AS,Kessler SW,Goff JP,etal.Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells[J].J Immunol, 1991, 146(5): 1410~1415
5 man ES. The role of mast cells in inflammatory responses in the lung[J].Crit Rev Immunol , 1993, 13: 36~70.
6 Church MK, Levi-Schaffer F. The human mast cell. J Allergy Clin Immunol. 1997 Feb;99(2):155
7 Egozi EI, Ferreira AM, Burns AL, et al. Mast cells modulate the inflammatory but not the proliferative response in healing wounds. Wound Repair Regen. 2003 ,11(1):46
8 Miller HR, Pemberton AD. Tissue-specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut. Immunology. 2002, 105:375
9 Pallaoro M, Fejzo MS, Shayesteh L, et al. Charac- terization of genes encoding known and novel human mast cell tryptases on chromosome 16p13.3. J Biol Chem. 1999,274(6):3355
10 Pereira PJ, Bergner A, Macedo-Ribeiro S, et al. Human beta-tryptase is a ring-like tetramer with active sites facing a central pore. Nature. 1998, 392(6673): 306
11 Dowdall JF, Winter DC, Baird AW, et al. Biological role and clinical implications of mast cells in surgery. Surgery. 2002,132(1):1
12 Akers IA, Parsons M, Hill MR, et al. Mast cell tryptase stimulates human lung fibroblast proliferation via protease-activated receptor-2. Am J Physiol Lung Cell Mol Physiol. 2000,278(1): L193
13 Abe M, Kurosawa M, Ishikawa O, et al. Effect of mast cell–derived mediators and mast cell–related neutral proteases on human dermal fibroblast proliferation and type I collagen production. J Allergy Clin Immunol. 2000 ,106(1 Pt 2):S78
14 Garbuzenko E, Nagler A, Pickholtz D, et al. Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis. Clin Exp Allergy. 2002 Feb; 32(2): 237
15 Mirza H, Schmidt VA, Derian CK, et al. Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell alpha- or beta-tryptases. Blood. 1997, 90 (10):3914
16 Compton SJ, Cairns JA, Holgate ST, et al. The role of mast cell tryptase in regulating endothelial cell proliferation, cytokine release, and adhesion molecule expression: tryptase induces expression of mRNA for IL-1 beta and IL-8 and stimulates the selective release of IL-8 from human umbilical vein endothelial cells. J Immunol. 1998,161(4):1939
17 Sommerhoff CP. Mast Cell Tryptases and Airway Remodeling. Am J Respir Crit Care Med. 2001 Nov 15;164(10 Pt 2):S52
18 Sime PJ,O Reilly KM.Fibrosis of the lung and other tissues:New concepts in pathogenesis and treatment[J].Clin Immunol,2001,99(3):308
19 Lukacs NW,Hogaboam C,Chensue SW,et al. Type1/type2 cytokine paradigm and progression of pulmonary fibrosis[J].Chess, 2001,120(1Sup pl): 5s
20 Kunkel SL, Chensue SW, Lukacs N,et al. Cytokine phenotypes serve as a paradigms for experimental immune-mediated lung diseases and remodeling[J].Am J Respir Cell Mol Biol,2003,29(3 Suppl): s63
21 Wallace WA,Ramage EA,Lamb D,et al.A type2(Th2-type pattern of response predominates in the pulmonary interstitium of patients with cryptogenic fibrosing alveolitis (CEA).Clin Exp Immunol,1995,101: 436~441
22 Furuie H,Yamasaki H,Suga M,etal.Altered accessory cell function of alverlar macrophages:Apossible mechanism for Induction of Th2 secretory profile in idiopathic pulmonary fibrosis.Eur RespirJ,1997;10:787~794
1 Burgess HA, Daugherty LE, et al. PPAR-γagonists inhibit TGF- β induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis[J].Lung Cell Mol Physiol ,2005,288: 1146~1153
2 Coker R K,Laurent G J.Pulmonary fibrosis:cytokines in the balance[J].Eur Respir J,1998,11:1218
3 Roberts A B, Piek E,Bottinger E P,et al.Is Smad3 a major player in singal transduction pathways leading to fibrogenesis?[J]Chest,2001,120(1 Suppl):43s
4 Gauldie J,Galt T,Bonniaud P,et al.Transfer of the active form of transforming growth factor-beta 1 gene to newborn rat lung induces changes consistent with bronchopulmonary dysplasia[J].Am J Pathol, 2003,163 (6):2575
5 Kasper M,Seidel D,Knels L,et al.Early signs of lung fibrosis after in vitro treatment of rat lung slices with CdCI(2) and TGF-beta(1)[J].Histochem Cell Biol,2004, 121(2):131
6 Oemar BS,Luscher TF.Connective tissue growth factor, friend or foe?[J].Arterioscler Thromb VascBiol, 1997, 17 (8):1483~1489
7 Moussad EE, Rrigstock DR.Connective tissue growth factor; what’s in a name? Mol Genet Metab, 2000, 7; 276~292
8 Williams EJ, Gaca MD, Brigstock DR, et al. Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells. J Hepatol, 2000,32(5):754~61
9 Leask A, Sa S, Holmes A, et al. The control of ccn2 (ctgf) gene expression in normal and scleroderma fibroblasts. Mol. Pathol. 2001, 54:180~183
10 Joseph A, Lasky, Luis A, et al .Connective tissue growth factor mRNA expression is upregulated in bleomycin- induced lung fibrosis[J]. Am J Physiol Lung Cell Mol Physiol ,1998,275(2): 365~371
11 Crean JKG,Finlay D,Murphy M,etal.The role of p42/p44 MAPK and protein kinase B in connective tissue growth factor induced extracellular matrix protein production,cell migration, and actin cytoskeletal rearrangement in human mesangial cells [J].J Biol Chem,2002, 277(46): 44187~ 44194
12 Chen MM, Ian A, Abraham,et al.CTGF expression is induced by TGF-beta in cardiac fibroblasts and cardiac myocytes: a potential role heart fibrosis.J Mot Cdl Cndio1,2000,32:180
13 Hu B , Wu Z , Phan SH. Smad3 mediates transforming growth factor-beta-induced alpha-smooth muscle actin expression. Am J Respir Cell Mol Biol. 2003,29(3 Pt 1):397~404
14 Gu L , Zhu YJ,Yang X, et al. Effect of TGF-beta/Smad signaling pathway on lung myofibroblast differentiation. Acta Pharmacol Sin. 2007,28(3):382~91
15 Folger PA, Zekaria D, Grotendorst G, Masur SK. Transforming growth factor-beta-stimulated connective tissue growth factor expression during corneal myo- fibroblast differentiation. Invest Ophthalmol Vis Sci. 2001 ,42(11):2534~41
16 Huang HC,Yang M, Li JZ, Wang HY. Connective tissue growth factor promotes the proliferation of myofibroblast through Erk-1/2 signaling pathway Zhonghua Yi Xue Za Zhi. 2005 , 25;85(19):1322~6
17 Wu SH, Wu XH, Lu C, et al. Lipoxin A4 inhibits proliferation of human lung fibroblasts induced by connective tissue growth factor. Respir Cell Mol Biol. 2006,34(1):65~72
18 Sanlana A,Saxena B,Nancy A,etal.Increased expression of transforming growth factorβ isoforms(β1、β2、β3 ) in bleomycin-induced pulmonary fibrosis.Am Respir Cell Mol Biol ,1995 ,13 ; 34~44
19 Joseph A. Lasky, Luis A, etal. Connective tissue growth factor mRNA expression is upregulated in bleomycin- induced lung fibrosis. Am J Physiol Lung Cell Mol Physiol. 1998,275: 365~371
20 张雷,卢惠苹,赖国祥.结缔组织生长因子在博莱霉素诱导的小鼠肺纤维化中的作用探讨.细胞与分子免疫学杂志,2005,21;290~292
21 Broekelmann TJ ,Limper AH ,Colby TV,et al. Transforming growth factors β1 is present at sites of extracellrar matrix gene expression in human pulmonary fibrosis.Prnc Natl .Acad Sci USA,1991,88:6642~6646
22 Pan LH,Yamauchi K,Uzuki M,et al.Type Ⅱ alveolar epithelial cells and interstitial fibroblasts express connective tissue growth factor in IPF[J]. Eur Repir J,2001,17(6):1220~1227
23 Hamada H,Vallyathan V,Cool CD,et al.Mast cell basic fibroblast growth factor in silicosis[J].Am J Respir Crit Care Med,2000,161:2026
1 Oemar BS,Luscher TF.Connective tissue growth factor, friend or foe?[J].Arterioscler Thromb Vasc Biol, 1997, 17 (8):1483~1489
2 BrigstockDR.The connective tissue growth factor/ cysteine-rich 61/nephroblastoma overexpressed (CCN) family[J].Endocr Rev 1999,20:189
3 Kubota S,Hattori T,Nakanishi T,etal.Involvement of cisacting repressive element(s) in the 3’-untranslated region of human connective tissue growth factor gene.FEB Lett,1999,450(1-2):84~88
4 Bradham DM,Igarashi A,PotterRL,etal.Connective tissue growth factor:acysteine-rich mitogen secreted by human vascuclar endothelial cells is related to the SRC-induced immediate early gene product CEF-10[J].J Cell Biol,1991, 114(6):1285~1294
5 Murray RJ,McDonald NQ,Blundell TL,etal.Topological similaritie in TGF-beta,PDGF and NGF define a super-family of polypeptide growth factors.Structure 1993, 1:153~159
6 Gupta S,Clarkson MR,Duggan J,etal.Brady Connective tissue growth factor:protential role in glomerulosclerosis and tubulointer-stitial fibrosis.Kidney Inter,2000, 58(5): 1389~1399
7 Grotendorst GR.Connective tissue growth factor:amediator of TGF-beta action on fibroblasts[J].Cytokine Growth Factor Rev, 1997,8:171
8 Chen MM, Ian A, Abraham,et al.CTGF expression is induced by TGF-beta in cardiac fibroblasts and cardiac myocytes: a potential role heart fibrosis.J Mot Cdl Cndio1,2000.32:180
9 Duncan MR, Frazier KS, Abremaon S et al. Camective tissue growth factor mediates .transforming growth factor beta-induced collagen synthesis:Down-regulation CAMP. FASEB J,1999,13; 1774
10 Grotendorst GR, Okochi H, Hayashi N. A novel trans-forming growth factor beta response element controls the expression of the connective tissue growth factor gene.Cell Growth Differ, 1996, 7(4):469~480
11 Leask A, Sa S, Holmes A, Shiwen X, et al. The control of ccn2 (CTGF) gene expression in normal and scleroderma fibroblasts. Mol Pathol, 2001,54(3):180~183
12 Chambers RC, Leoni P, Blanc-Brude OP, et al. Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease- activated receptor-1. J Biol Chem 2000;275:35584~91
13 Abraham DJ, Shiwen X, Black CM, et al. Tumor necrosis factor alpha suppresses the induction of connective tissue growth factor by transforming growth factor-beta in normal and scleroderma fibroblasts. J Biol Chem 2000; 275: 15220 ~5
14 Pendurhti UR, Allen KE, Ezban M, et al. Factor VIIA and thrombin induce the expression of cyr61 and connective tissue growth factor, extracellular matrix signaling proteins that could act as possible downstream mediators in factor VIIA x tissue factor-induced signal transduction. J Biol Chem 2000; 275: 14632~41
15 Riser BL, Denichilo M, Cortes P, et al. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol 2000;11:25~38
16 Murphy M, Godson C, Cannon S, et al. Suppression subtractive hybridization identified high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J Biol Chem 1999;274:5830~4
17 Dammeier J, Beer HD, Brauchle M, et al. Dexamethason is a novel potent inducer of connective tissue growth factor expression. Implications for glucocorticoid therapy.J Biol Chem 1998; 273: 18185~90
18 Frazier K,Williams S,Kothapalli D,et al.Stimulation of fibroblast cell growth, matrix production and granulation tissue formation by connective tissue growth factor.J Invest Dermatol,1996,107: 404~411.
19 Hahn A,Heusinger-Ribeiro J,Lanz T,et al.Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton.J Biol Chem,2000,275(48):37429~35
20 Yokoi H,Mukoyama M,Sugawara A,etal.Role of Connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis.Am J Physiol Renal Physiol,2002,282(5):933~942
21 Luo Z, Li H, Zhang J, etal. Effects of human connective tissue growth factor gene transfection on migration of human umbilical vein endothelial cell. Clin Hemorheol Microcirc.2006;34(1-2):185~92
22 David R,Brigstock.The Connective tissue growth factor/ Cysteine-Rich61/Nephroblastoma overexpressed (CCN) family. Endo Reviewe,1999,20(2):189~206
23 Hishikawa K,Oemar BS,Nakaki T.Static pressure regulates Connective tissue growth factor expression in human mesangial cells.J Biol Chem,2001,276(20):16797~16803
24 Shi-wen X, Stanton LA, Kennedy L,etal. CCN2 is necessary for adhesive responses to transforming growth factor-beta1 in embryonic fibroblasts.J Biol Chem.2006, 281(16):10715~26
25 Chen CC,Chen N,Lau LF.The Angiogenic Factors Cyr61 and Connective Tissue Growth Factor Induce Adhesive Signaling in Primary Human Skin Fibroblasts[J].J Biol Chem,2001,276(13):10443~10452
26 Jedsadayanmata A,Chen CC,Kireeva ML,etal.Activation dependent adhesion of human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is mediated through integrin alpha(IIb)beta(3)[J].J biol Chem,1999, 274 (34) :24321~24327
27 Luo Z, Li H, Zhang J, etal. Effects of human connective tissue growth factor gene transfection on migration of human umbilical vein endothelial cell. Clin Hemorheol Microcirc.2006;34(1-2):185~92
28 Lasky JA, Ortiz LA, Tonthat B, et al. Connective tissue growth factor mRNA expression is upregulated in bleomycin-induced lung fibrosis. Am J Physiol, 1998, 275(2 Pt 1):365~371
29 Ricupero DA, Rishikof DC, Kuang PP, et al. Regulation of connective tissue growth factor expression by prostaglandin E(2). Am J Physiol, 1999,277(6Pt1): 1165 ~1171
30 Ricupero DA, Romero JR, Rishikof DC, et al. Des- Arg(10)- kallidin engagement of the B1 receptor stimulates type I collagen synthesis via stabilization of connective tissue growth factor mRNA[J]. J Biol Chem, 2000, 275(17):12475~12480
31 Xiao R, Liu FY, Luo JY,etal.Effect of small interfering RNA on the expression of connective tissue growth factor and type I and III collagen in skin fibroblasts of patients with systemic sclerosis. Br J Dermatol. 2006, 155 (6): 1145~53
32 Folger PA, Zekaria D, Grotendorst G, Masur SK.Transforming growth factor-beta-stimulated connective tissue growth factor expression during corneal myofibroblast differentiation. Invest Ophthalmol Vis Sci. 2001,42(11):2534~41
33 Gu L,Zhu YJ,Yang X, et al.Effect of TGF-beta/Smad signaling pathway on lung myofibroblast differentiation. Acta Pharmacol Sin. 2007,28(3):382~91
34 Folger PA, Zekaria D, Grotendorst G, Masur SK. Transforming growth factor-beta-stimulated connective tissue growth factor expression during corneal myo- fibroblast differentiation. Invest Ophthalmol Vis Sci. 2001,42(11):2534~41
35 Huang HC,Yang M, Li JZ, Wang HY. Connective tissue growth factor promotes the proliferation of myofibroblast through Erk-1/2 signaling pathway. Zhonghua Yi Xue Za Zhi. 2005,25;85(19):1322~6
36 杨 敏,黄海长,李惊子等.结缔组织生长因子增加 TGFB活 化 成 肌 纤 维 细 胞 的 作 用 . 基 础 医 学 与 临 床 . 2005,25(1)25~29
37 Wu SH, Wu XH, Lu C, et al. Lipoxin A4 inhibits proliferation of human lung fibroblasts induced by connective tissue growth factor. Respir Cell Mol Biol. 2006,34(1):65~72
38 黄山英,宋良文,张勇等.CTGF 表达在大鼠放射性肺纤维 化 中 的 作 用 . 中 华 放 射 医 学 与 防 护 杂 志 .2005, 25(3);213~216