博莱霉素致肺纤维化中TGF-β1表达

25只大鼠随机分为正常对照组和灌注博莱霉素实验组。在 3d ,7d ,14d ,30d分别取肺组织 ,以免疫组织化学技术测定TGF β1蛋白表达。结果显示未经博莱霉素处理正常对照组大鼠的少数细支气管粘膜上皮细胞的胞浆有TGF β1蛋白弱表达 ;博莱霉素处理大鼠的支气管、细支气管粘膜上皮细胞则TGF β1表达更明显和范围更广 ;特别是在 3～ 7d肺泡腔和间质中出现大量的TGF β1强阳性的肺泡巨噬细胞 ,14～ 30d时在肺泡腔和间质中只有少量TGF β1阳性的肺泡巨噬细胞。提示TGF β1蛋白表达阳性的肺泡巨噬细胞在急性肺泡炎期明显增加 ,对肺纤维化的发生和发展可能起重要的作用     

肝细胞生长因子抑制MRC-5成纤维细胞所致的纤维化作用

本研究采用人胎儿肺来源的成纤维细胞株MRC－5，作为体外研究肝细胞生长因子（HGF）抗组织纤维化的模型。成纤维细胞MRC－5被转化生长因子β1（TGF－β1）转化为肌成纤维细胞后，应用外源人重组HGF修饰细胞，检测细胞中分子表达水平的变化。研究发现，TGF－β1在诱导成纤维细胞株MRC－5转化为肌成纤维细胞的同时，上调HGF受体/c-Met在肌成纤维细胞中的表达。而且，应用外源人重组HGF修饰细胞后，内源性TGF－β1的表达水平被降低，内源性HGF的表达水平上调，基质金属蛋白酶I（MMP－I）mRNA表达增加，从而加速了细胞外基质主要成分－胶原蛋白I[Col(I)]的水解。结果显示在组织纤维经过程中，HGF通过其受体c-Met直接作用于肌成纤维细胞，抑制细胞内TGF－β1的表达，在一定程度上抑制组织纤维化的发生和发展。研究中我们还发现，肝素结合表皮生长因子样生长因子（HB－EGF）上调肌成纤维细胞中HGF的表达，是其抑制肌成纤维细胞形成、抑制组织纤维化的可能机制之一。     

热休克蛋白47参与TGF-β1促人胚肺细胞胶原合成

  摘要：目的 观察热休克蛋白47（HSP47）对人胚肺成纤维细胞（HELF）胶原合成及间质细胞标志物的影响,探讨它与肺纤维化的关系。方法 用TGF－β1诱导HELF表达HSP47,观察用5ng/mL TGF-β1作用不同时间和用不同浓度TGF－β1作用于HELF48h; WST法检测TGF-β1对HELF增殖影响;细胞免疫荧光法检测HSP47的表达;免疫印迹技术检测HSP47、Ⅰ型胶原（COLLAGENⅠ）及间质细胞标志物α-平滑肌肌动蛋白(α-SMA)、波形蛋白(VIMENTIN)的表达。结果 正常HELF表达少量HSP47,随着HELF在TGF-β1作用时间的延长和剂量逐渐增加,HSP47、COLLAGENⅠ、α-SMA和VIMENTIN的表达均同步增强（P<0.05）。结论 HSP47在TGF－β1的作用下,促进人胚肺成纤维细胞胶原蛋白的合成,作为胶原特异性分子伴侣在肺纤维化的发生发展过程中发挥至关重要的作用。     

TGF-β1对人肾间质成纤维细胞PAI-1表达的作用

目的：探讨转化生长因子β1（TGF－β1）在肾间质纤维化中的作用机理。方法：体外分离培养人肾间质成纤维细胞,应用不同浓度（0,2,5,10ug/L)的TGF－β1培养刺激24h,和以5μg/L TGF－β1作用不同时间（0,6,12,24,48h),以逆转录－PCR技术检测成纤维细胞表达纤溶酶原激活剂的抑制物（PAI－1）mRNA的变化。结果 TGF－β1 在0－10μg/L浓度范围可促进肾间质成纤维细胞2PAI－1 mR-NA的表达,且呈剂量效应相关性,在0－48h内,5μg/L TGF－β1刺激后PAI－1表达,参与肾纤维化中基质的合成和降解过程,从而在肾间质纤维化过程中发挥作用。     

芪丹颗粒剂对大鼠肺纤维化模型的干预作用及对TGF-β1、TNF-α表达的影响

目的：探讨芪丹颗粒剂对博莱霉素A5所致大鼠肺纤维化的干预作用及可能的机制。 方法： SD大鼠经气管内一次性灌注博莱霉素A5(5 mg/kg)诱导肺纤维化，随后分别每日给予芪丹颗粒剂灌胃（芪丹组，3 125 mg/kg）、氢化可的松腹腔注射（氢可组，25 mg/kg）进行干预。对照组气管内灌注和灌胃均用生理盐水。各组动物均于药物干预后第7、14、28 d分别处死。用苏木素-伊红（HE）评价肺组织病理学变化、免疫组化（SABC法）测定肺组织TGF-β1、TNF-α蛋白的表达。 结果： 芪丹组肺泡炎及肺纤维化程度均明显轻于模型组和氢可组，TGF-β1、TNF-α蛋白的表达亦显著低于模型组和氢可组（P<0.01）。 结论： 芪丹颗粒剂可明显减轻博莱霉素A5诱导的大鼠肺纤维化的程度, 其作用机制可能部分通过降低TGF-β1、TNF-α蛋白的表达而实现。     

肺泡Ⅱ型TGFβ1和PDGF基因表达及其肺纤维化中的意义

目的：研究转化生长因子β1（TGFβ1）和血小板源性生长因子（PDGF）在肺泡Ⅱ型细胞中的表达及其在肺纤维化过程中的意义。方法：分离培养正常成年大鼠肺泡Ⅱ型细胞,建立大鼠矽肺模型,用原位杂交和免疫组化技术检测体外培养的和矽肺病变中的肺泡Ⅱ型细胞TGFβ1和PDGF-B mRNA和蛋白的表达。结果：（1）体外培养的肺泡Ⅱ型细胞免疫组化染色TGFβ1强阳性,PDGF-B弱阳性;原位杂交TGFβ1和PDGF-B mRNA均为阳性。（2）大鼠矽肺实验组增生的肺泡Ⅱ型细胞明显表达TGFβ1和PDGF-B mRNA和蛋白;对照组仅有部分正常肺泡Ⅱ型细胞TGFβ1 mRNA呈弱阳性。结论：增生的肺泡Ⅱ型细胞有TGFβ1和PDGF-B基因表达,其在矽肺纤维化病变中可能起重要作用。     

干扰素γ对实验性大鼠肺纤维化、肺组织干扰素γ和转化生长因子β表达的影响

目的：研究干扰素γ（IFN-γ）对博莱霉素诱导的大鼠肺纤维化实验模型肺泡炎和肺纤维化的影响,并探讨其对肺纤维化影响的可能机制。方法：60只SD大鼠随机分为正常对照组（N组）、模型对照组（M组）和干扰素γ组（R组）,每组各20只大鼠。于造模后3、7、14、28天分批处死动物,留取肺组织,观察肺泡炎和肺纤维化的程度,测定肺组织中的羟脯氨酸含量、IFN-γmRNA、转化生长因子β（TGF-β）mRNA的表达。结果：M组、R组大鼠肺泡炎3、7、14、28天均较N组严重,7天时最重,R组14天时肺泡炎显著高于M组;M组、R组肺纤维化评分及肺羟脯氨酸含量14、28天均高于N组,于28天最重;R组肺组织IFN-γmRNA表达3、7、14、28天均显著高于M组;R组肺组织中TGF-βmRNA表达在3天显著高于M组（P〈0.05）。结论：在博莱霉素诱导大鼠肺纤维化早期给予干扰素γ并不能减轻肺部炎症和肺纤维化,可能与其促进肺组织IFN-γ、TGF-β基因表达有关。     

当归注射液及前列腺素E2对结缔组织生长因子在大鼠肺纤维化模型中表达作用的比较研究

肺间质纤维化是多种病因引起的异质性疾病，其主要病理特点是早期的弥漫性肺泡炎和后期大量成纤维细胞的病理性增生及基质胶原进行性积聚并取代正常的肺组织结构。临床缺乏有效治疗手段。本文采用博莱霉素大鼠模型观察结缔组织生长因子（CTGF）mRNA的表达及其与纤维连结蛋白（Fibronection，FN）、胶原蛋白Ⅲ（Collegen Ⅲ，Col-Ⅲ）及肺纤维化程度的关系，并用25％当归注射液、前列腺素E2（dmPGE2）进行治疗，观察它们抗肺纤维化作用的可能机制。     

甘草酸对博莱霉素诱导的实验性肺纤维化的干预作用

目的:探讨甘草酸(GA)对博莱霉素(BLM)诱导的小鼠肺纤维化的干预作用及其可能机制。方法:将160只雄性C57BL/6J小鼠随机分为生理盐水(NS)组、BLM组、BLM+NS组和BLM+GA组。通过口咽气管吸入法吸入博莱霉素(2.5 mg/kg)建立实验性肺纤维化模型,BLM+GA组及BLM+NS组每天给予40 mg/kg甘草酸或等体积的生理盐水灌胃,于术后第3、7、14、21天取材。采用HE染色和Masson染色观察肺组织病理学变化及纤维化程度,采用流式细胞术检测循环单核细胞和肺泡巨噬细胞的亚群比例变化,采用RT-qPCR检测肺组织中转化生长因子β1(TGF-β1)mRNA的表达水平,采用碱水解法检测肺组织中羟脯氨酸(HYP)含量。结果:与NS组相比,BLM组和BLM+NS组肺组织的炎症浸润及胶原含量明显增多,实验性肺纤维化模型制备成功。与BLM+NS组相比:BLM+GA组肺组织的炎症细胞浸润及胶原纤维沉积较少;第3、7天的Ly6C~(hi)单核细胞亚群比例和第7、14天肺泡巨噬细胞M2表型比例显著降低(P0.01);肺组织TGF-β1 mRNA表达量和HYP含量显著降低(P0.01)。结论:GA可以减轻博莱霉素诱导的小鼠肺组织的炎症反应和胶原纤维沉积,可能与GA对单核巨噬细胞的表型偏移和调控以及肺组织TGF-β1的表达下调有关。     

甘草甜素减轻博莱霉素致大鼠肺纤维化

目的探讨甘草甜素减轻博莱霉素致大鼠肺纤维化的机制。方法将大鼠随机分成对照组、博莱霉素致大鼠肺纤维化模型组、地塞米松(DXM)、甘草甜素低、中及高剂量治疗组。注博莱霉素后第28天,连续14 d同一时间段腹腔注射。第15天取左肺下叶供免疫组化检测TGF-β1、IFN-γ表达;右肺下叶供病理组织观察。用ELISA双抗体夹心法测定各组大鼠血清IL-4、IFN-γ含量。结果 (1)模型组大鼠肺组织成纤维细胞反应增强,TGF-β1染色及定量表达增强;而甘草甜素各组、DXM组减轻明显(P<0.01);(2)模型组大鼠血清IL-4含量较高,IFN-γ含量较低;甘草甜素各组及DXM组则相反(P<0.05)。(3)甘草甜素各组、DXM组肺组织IFN-γ染色及定量表达增强,甘草甜素低剂量组较其他组增强(P<0.01)。结论甘草甜素通过降低肺组织TGF-β1表达和血清IL-4含量,上调IFN-γ表达,提高血清IFN-γ含量,从而减轻肺纤维化程度。     

实验性肺纤维化大鼠组织整合素α5β1和转化生长因子—β的表达

目的 研究纤维连接蛋白受体整合蛋白受体整合素α５β１在大鼠肺纤维化中的作用。方法 用免疫组化方法观察实验性大鼠肺纤维化纤维连接蛋白及其受体整合素α５β１和转化生长因子－β表达的动态变化：用Ｎｏｒｔｈｅｍ印迹杂交和免疫细胞化学方法观察ＴＧＦ－β对体外培养的大鼠肺成纤维细胞整合素α５β１ ｍＲＮＡ和 白表达的影响。结果 （１）实验组１￣３天,病灶内上皮细胞和骨皮细胞整合素α５β１表达明显增强,炎细胞及     

Increased expression of epimorphin in bleomycin-induced pulmonary fibrosis in mice

Epimorphin was originally identified as a mesenchymal, cell surface-associated protein that modulates epithelial morphogenesis in embryonic organs, whereas pulmonary fibrosis is a process of wound healing, which in part mimics the process of fetal lung development. We investigated the temporal and spatial changes in the distribution of epimorphin protein and expression of its messenger RNA (mRNA) in bleomycin-induced pulmonary fibrosis in mice. Immunohistochemical analysis showed that low levels of epimorphin were present in the bronchiolar, alveolar, and vascular walls of normal adult lungs. However, from Day 7 until Day 28 after bleomycin treatment, increasing levels of epimorphin immunoreactivity were detected in the mesenchymal cells and in the extracellular matrix within intra-alveolar fibrotic lesions. Moreover, Northern blots showed corresponding increases in epimorphin mRNA expression. Re-epithelialization of epimorphin-rich intra-alveolar fibrosis was complete by Day 28 after bleomycin, and by Day 56, epimorphin immunoreactivity had declined. In situ hybridization and confocal microscopic studies confirmed expression of epimorphin mRNA by mesenchymal cells situated within early fibrotic lesions, whereas immunoelectron microscopy localized the epimorphin to the endoplasmic reticulum of the mesenchymal cells and to the basement membrane and collagen fibrils in the area. These results suggest that epimorphin may contribute to the remodeling of pulmonary fibrosis via epithelial-mesenchymal interactions.     

TGF‐beta1 and TGFBR1 are Expressed in Ameloblasts and Promote MMP20 Expression

Transforming growth factor‐beta (TGF‐beta) signaling exerts a wide spectrum of biological functions. To investigate TGF‐beta signaling in amelogenesis, we initially assessed the expression of TGF‐beta1 and TGF‐beta receptor 1 (TGFBR1) in developing teeth by immunohistochemistry. Both TGF‐beta1 and TGFBR1 were strongly expressed in secreting ameloblasts. Next, we studied the effects of TGF‐beta signaling on the expression of MMP20 and KLK4 mRNA using ameloblast‐lineage cells (ALC) in vitro. Our RT‐PCR study showed that TGF‐beta1, TGFBR1, and enamel matrix proteases (MMP20 and KLK4) were expressed in ALC. Following TGF‐beta1 treatment, the expression of MMP20 mRNA, but not KLK4 mRNA, was significantly upregulated. To further confirm the TGF‐beta signaling involvement in the MMP20 expression, we constructed the activated TGFBR1 vector and transfected the construct into ALC. The activated TGFBR1 notably promoted MMP20 expression, but had no obvious effects on the KLK4 mRNA expression. Our studies strongly suggest that TGF‐beta signaling involved in amelogenesis is partially mediated by regulating the expression of MMP20 mRNA. Anat Rec, 292:885–890, 2009. © 2009 Wiley‐Liss, Inc.     

Caveolin-1 deficiency protects from pulmonary fibrosis by modulating epithelial cell senescence in mice

Idiopathic pulmonary fibrosis is associated with a decreased expression of caveolin-1 (cav-1), yet its role remains unclear. To investigate the role of cav-1, we induced pulmonary fibrosis in wild-type (WT) and cav-1-deficient (cav-1(-/-)) mice using intratracheal instillation of bleomycin. Contrary to expectations, significantly less collagen deposition was measured in tissue from cav-1(-/-) mice than in their WT counterparts, consistent with reduced mRNA expression of procollagen1a2 and procollagen3a1. Moreover, cav-1(-/-) mice demonstrated 77% less α-smooth muscle actin staining, suggesting reduced mesenchymal cell activation. Levels of pulmonary injury, assessed by tenascin-C mRNA expression and CD44v10 detection, were significantly increased at Day 21 after injury in WT mice, an effect significantly attenuated in cav-1(-/-) mice. The apparent protective effect against bleomycin-induced fibrosis in cav-1(-/-) mice was attributed to reduce cellular senescence and apoptosis in cav-1(-/-) epithelial cells during the early phase of lung injury. Reduced matrix metalloproteinase (MMP)-2 and MMP-9 expressions indicated a low profile of senescence-associated secretory phenotype (SASP) in the bleomycin-injured cav-1(-/-) mice. However, IL-6 and macrophage inflammatory protein 2 were increased in WT and cav-1(-/-) mice after bleomycin challenge, suggesting that bleomycin-induced inflammatory response substantiated the SASP pool. Thus, loss of cav-1 attenuates early injury response to bleomycin by limiting stress-induced cellular senescence/apoptosis in epithelial cells. In contrast, decreased cav-1 expression promotes fibroblast activation and collagen deposition, effects that may be relevant in later stages of reparative response. Hence, therapeutic strategies to modulate the expression of cav-1 should take into account cell-specific effects in the regenerative responses of the lung epithelium to injury.     

当归注射液及前列腺素E_2对结缔组织生长因子在大鼠肺纤维化模型中表达作用的比较研究

目的探讨结缔组织生长因子(CTGF)在大鼠肺间质纤维化中的表达及其在25%当归注射液、前列腺素E2(dmPGE2)的肺脏保护作用中的意义。方法SD大鼠随机分为对照组、模型组、当归组、PGE2组。按博莱霉素(BLM)5mg/kg体重复制大鼠肺纤维化模型,当归组、dmPGE2组术后分别给于25%当归注射液(腹腔内注射)、dmPGE2(肌肉注射),于第28天处死全部大鼠。制备大鼠肺组织切片,用原位杂交及免疫组化SABC法分别检测CTGF mRNA、纤维连结蛋白(FN)和Ⅲ型胶原(Col-Ⅲ)在肺内的表达,HE染色评价肺纤维化程度。结果模型组CTGF mRNA、FN和Col-Ⅲ的表达及肺纤维化程度明显高于对照组(P<0.01),而当归组及PGE2组明显低于模型组(P<0.01)。两治疗组间比较,除CTGF外,其余指标无显著性差异(P>0.05),各组指标的相关分析表明,CTGF表达水平与肺纤维化程度(r=0.886;P<0.01)、FN(r=0.836;P<0.01)和Col-Ⅲ(r=0.918;P<0.01)呈正相关关系。结论25%当归注射液可能通过下调CTGF而减轻肺间质纤维化。     

大黄酸通过抑制miR-21而干预TGF-β1/Smad通路并减轻博莱霉素所致大鼠肺纤维化

目的:观察大黄酸(rhein,RH)对博莱霉素所致肺纤维化大鼠微小RNA-21(miR-21)表达以及转化生长因子β1(TGF-β1)/Smad通路的影响。方法:博莱霉素一次性气管内注射复制大鼠肺纤维化模型,随机分为RH低、中、高剂量组及模型(model)组;正常对照组大鼠气管内注射生理盐水。用药28 d后,HE染色观察各组大鼠肺组织形态学的变化;测定肺系数、肺组织羟脯氨酸含量;real-time PCR检测肺组织中miR-21和TGF-β1/Smad7m RNA表达;Western blot法分析TGF-β1和Smad7蛋白的表达。结果:与model组相比,RH用药组大鼠的肺泡炎及肺纤维化程度有明显降低,肺系数及肺组织羟脯氨酸含量也显著减少,肺组织中miR-21表达下降,TGF-β1的m RNA和蛋白表达水平也明显下降,Smad7的mRNA及蛋白表达水平明显增高(P0.05)。结论:RH抗肺纤维化的作用可能与抑制miR-21的表达,从而干预TGF-β1/Smad信号通路,减少细胞外基质沉积有关。     

Receptor-activated Smad localisation in bleomycin-induced pulmonary fibrosis

BACKGROUND: Recent advances in fibrosis biology have identified transforming growth factor (TGF)-beta type I receptor-mediated activation of Smads as playing a central part in the development of fibrosis. However, to date, there have been few studies that examined the localisation and distribution of receptor-activated Smads protein (R-Smads: Smad2 and 3) during the fibrosis progression. AIMS: To histopathologically assess the time-course change of the localisation and distribution of the Smads protein in pulmonary fibrosis. METHODS: Pulmonary fibrosis was induced by intranasal injection of bleomycin (0.3 U/mouse). Lungs were isolated 2, 5, 7, 9 and 14 days after bleomycin treatment. Histological changes in the lungs were evaluated by haematoxylin-eosin stain or Masson's trichrome stain, and scored. TGF-beta1, Smad3 and phosphorylated Smad2 localisations in lung tissues were determined by immunohistochemistry. RESULTS: The bleomycin treatment led to considerable pulmonary fibrotic changes accompanied by marked increase in TGF-beta1 expression in infiltrating macrophages. With the progression in fibrosis (day 7-14), marked increases in Smad3-positive and pSmad2-positive cells were observed. There were intense Smad3-positive and pSmad2-positive signals localised to the nuclei of the infiltrating macrophages and to type II epithelial cells, and less intense signals in fibroblasts and hyperplastic alveolar/bronchiolar epithelial cells. CONCLUSIONS: The time-course data of TGF-beta1 and R-Smads indicate that progressive enhancement of TGF-beta1 signalling via R-Smad is activated in the process of fibrosis progression.     

Role of the chemokine receptor CXCR2 in bleomycin-induced pulmonary inflammation and fibrosis

Pulmonary fibrosis is characterized by chronic inflammation and excessive collagen deposition. Neutrophils are thought to be involved in the pathogenesis of lung fibrosis. We hypothesized that CXCR2-mediated neutrophil recruitment is essential for the cascade of events leading to bleomycin-induced pulmonary fibrosis. CXCL1/KC was detected as early as 6 hours after bleomycin instillation and returned to basal levels after Day 8. Neutrophils were detected in bronchoalveolar lavage and interstitium from 12 hours and peaked at Day 8 after instillation. Treatment with the CXCR2 receptor antagonist, DF2162, reduced airway neutrophil transmigration but led to an increase of neutrophils in lung parenchyma. There was a significant reduction in IL-13, IL-10, CCL5/RANTES, and active transforming growth factor (TGF)-beta(1) levels, but not on IFN-gamma and total TGF-beta(1,) and enhanced granulocyte macrophage-colony-stimulating factor production in DF2162-treated animals. Notably, treatment with the CXCR2 antagonist led to an improvement of the lung pathology and reduced collagen deposition. Using a therapeutic schedule, DF2162 administered from Days 8 to 16 after bleomycin reduced pulmonary fibrosis and levels of active TGF-beta(1) and IL-13. DF2162 treatment reduced bleomycin-induced expression of von Willebrand Factor, a marker of angiogenesis, in the lung. In vitro, DF2162 reduced the angiogenic activity of IL-8 on human umbilical vein endothelial cells. In conclusion, we show that CXCR2 plays an important role in mediating fibrosis after bleomycin instillation. The compound blocks angiogenesis and the production of pro-angiogenic cytokines, and decreases IL-8-induced endothelial cell activation. An effect on neutrophils does not appear to account for the major effects of the blockade of CXCR2 in the system.