转化生长因子－β对人胚肺成纤维细胞胶原表达的影响

转化生长因子－β（ＴＧＦ－β）是一族多功能的细胞因子，具有调节细胞外基质代谢的功能，在肺间质纤维化的形成中具有重要作用。胚原是细胞外基质的主要成分。胶原积聚是肺间质纤维化的一个重要特征。为了进一步探讨肺间质纤维化形成的机制，在体外观察了ＴＧＦ－β对人胚胎肺成纤维细胞胶原蛋白合成及胶原Ｉ、胶原ＩＩｌｍＲＮＡ表达的影响。结果发现：ＴＧＦ－β能刺激胶原蛋白的合成，胶原Ｉ、胶原ＩＩＩｍＲＮＡ的表达。而对成纤维细胞的增殖影响不明显。因此认为ＴＧＦ－β可能与肺间质纤维化时胶原积聚有关。ＴＧＦ－β胶原合成的调节是通过ｍＲＮＡ水平。     

转化生长因子—β对人胚肺成纤维细胞胶原表达的影响

转化生长因子－β（ＴＧＦ－β）是一族多功能的细胞因子，具有调节细胞外基质代谢的功能，在肺间质纤维化的形成中具有重要作用。胶原是细胞外基质的主要成分。胶原积聚是肺间质纤维化的一个重要特征。为了进一步探讨肺间质纤维化形成的机制，在体外观察了ＴＧＦ－β对人胚胎成纤维细胞胶原蛋白合成及胶原Ｉ、胶原ＩＩＩｍＲＮＡ表达的影响。结果发现：ＴＧＦ－β能刺激胶原蛋白的合成，胶原Ｉ、胶原ＩＩＩｍＲＮＡ的表达。而对纤维     

转化生长因子β1与病毒性肝炎肝纤维化的关系

目的：研究转化生长因子β＿１（ＴＧＦ－β＿１）与病毒性肝炎肝纤维化发生的关系。方法：对５４例病毒性肝炎肝组织进行ＴＧＦ－β＿１以及α－平滑肌肌动蛋白（α－ＳＭＡ）、Ⅳ型胶原（Ⅳ－Ｃ）免疫组织化学研究，并检测其中４９例血清Ⅲ型前胶原肽（ＰⅢＰ）、层粘蛋白（ＬＮ）、透明质酸（ＨＡ）、Ⅳ－Ｃ等。结果：随着肝纤维化程度的加重，ＴＧＦ－β＿１在肝组织中的表达逐渐明显。在慢性肝炎重度、肝硬变慢性重型肝炎中的表达强度明显高于慢性肝炎中度、轻度及急性肝炎。同时ＴＧＦ－β＿１在肝组织中的表达与血清ＰⅢＰ、ＬＮ、ＨＡ、Ⅳ－Ｃ以及α－ＳＭＡ和Ⅳ－Ｃ在肝组织中的表达呈明显的正相关。结论：ＴＧＦ－β＿１通过刺激贮脂细胞，大量合成细胞外间质，最终导致肝脏的纤维化。     

细胞因子在特发性肺间质纤维化血管生成中的作用

目的 通过研究特发性肺间质纤维化（ＩＰＦ）患者开胸肺活检标本中胰岛素样生长因子（ＩＧＦ）－Ⅰ和血小板衍生长因子（ＰＤＧＦ）的表达,进一步阐明它们在ＩＰＦ过程中的作用。方法 采用免疫组化和原位杂交方法,分别利用ＩＧＦ－Ⅰ和ＰＤＧＦ的特异抗体和特异引物,检测其在ＩＰＦ患者开胸肺活检标本中的要布和表达。结果 在ＩＰＦ患者中,ＩＧＦ－Ⅰ主要分布在肺动脉血管、新生血管的平涌肌细胞和内皮细胞。肺泡巨噬细胞、Ⅱ     

转化生长因子β_1对纤维母细胞Ⅰ、Ⅲ型前胶原及胶原酶mRNA表达的影响

目的纤维母细胞是重要的胶原合成细胞，观察转化生长因子β１（ＴＧＦ－β１）对纤维母细胞Ⅰ、Ⅲ型前胶原及胶原酶ｍＲＮＡ表达的影响，以了解ＴＧＦ－β１在肝纤维化中的作用机制。方法首先对含有Ⅰ、Ⅲ型前胶原及胶原酶ｃＤＮＡ片段的质粒进行扩增、提纯及酶切，然后用自由引物延伸法进行地戈辛标记，并对探针进行检测；其次对纤维母细胞进行培养，加用ＴＧＦ－β１孵育后收集细胞并提取细胞总ＲＮＡ，最后将ＲＮＡ点样于尼龙膜上，并与上述探针进行斑点杂交。结果杂交结果显示ＴＧＦ－β能明显促进纤维母细胞Ⅰ型前胶原ｍＲＮＡ的表达（Ｐ＜０．０５），也能促进其Ⅲ型前胶原ｍＲＮＡ的表达（Ｐ＝０．０９）；而对其胶原酶ｍＲＮＡ的表达则起抑制作用（Ｐ＜０．０５）。结论ＴＧＦ－β可以从细胞及分子水平发挥其抗纤维化作用。     

原发性肝细胞癌患者转化生长因子β1的基因表达及临床意义

目的研究转化生长因子β１（ＴＧＦβ１）ｍＲＮＡ在原发性肝细胞癌患者中的表达及其临床意义。方法用ＲＴ－ＰＣＲ加ＤｏｔＢｌｏｔ法检测原发性肝细胞癌患者肝组织和外周血单个核细胞（ＰＢＭＣ）中ＴＧＦ－β１ｍＲＮＡ水平，并以正常肝组织和正常人的ＰＢＭＣ为对照。结果ＴＧＦ－β１ｍＲＮＡ水平在原发性肝细胞癌患者组肝组织（２．２２±０．８４，ｎ＝１６）和ＰＢＭＣ中（１．８３±１．２，ｎ＝２５）比较，差异无显著性（Ｐ＞０．０５），但两者均高于正常肝组织（０．９４±０．７６，ｎ＝５）和正常人的ＰＢＭＣ（０．６２±０．４０，ｎ＝１６）水平。结论ＴＧＦ－β１ｍＲＮＡ水平与原发性肝细胞癌有关，ＰＢＭＣ中ＴＧＦ－β１ｍＲＮＡ检测可望作为一项代替肝组织活检的指标，其表达水平与肝癌有关。     

肺间质病患者肺泡巨噬细胞蛋白激酶C活性的变化

目的 探讨肺间质纤维化时蛋白激酶Ｃ（ＰＫＣ）活性的变化。方法 应用放射生测定法检测９名健康者和１５例特发性肺间质纤维化９ＩＰＦ）患者支气管肺泡灌洗液（ＢＡＬＦ）中肺泡巨噬细胞（ＡＭ）的ＰＫＣ活性。结果 ＩＰＦ患者ＢＡＬＦ中ＡＭ总ＰＫＣ活性、南和胸膜ＰＫＣ活性均明显高于健康者（Ｐ〈０．０１和Ｐ〈０．０５）,ＡＭ的总ＰＫＣ活性与ＢＡＬＦ中细胞总数呈正相关（ｒ＝０．５９１７,Ｐ〈０．０５）。结论 ＰＫＣ     

间质性肺疾病支气管肺泡灌洗液的酶活性研究

目的探讨支气管肺泡灌洗液（ＢＡＬＦ）多项酶活性与间质性肺疾病（ＩＬＤ）的关系。方法检测３０例ＩＬＤｓ：包括特发性肺纤维化（ＩＰＦ）１８例和结节病（Ｓａｒｃ）１２例与９例正常对照者的ＢＡＬＦ中超氧化歧化酶（ＳＯＤ）、谷胱甘肽过氧化物酶（ＧＳＨ－ＰＸ）、血管紧张素转换酶（ＡＣＥ）和乳酸脱氢酶（ＬＤＨ）活性，并分类计数ＢＡＬＦ细胞成份。结果（１）ＩＰＦ组ＢＡＬＦ中各项酶活性均与对照组间差异有显著性（ＳＯＤ和ＧＳＨ－ＰＸ降低，ＡＣＥ和ＬＤＨ升高）（Ｐ＜０．０５）；而Ｓａｒｃ组仅见ＡＣＥ明显增高（Ｐ＜０．０５）。（２）ＢＡＬＦ－ＡＣＥ与Ｓａｒｃ组淋巴细胞百分比及ＣＤ＋４／ＣＤ＋８比值均有显著线性相关（Ｐ＜０．０５）。结论ＢＡＬＦ中ＳＯＤ、ＧＳＨ－ＰＸ、ＡＣＥ和ＬＤＨ活性测定，有助于进一步探讨ＩＬＤ发病机理和提供辅助诊断依据，ＢＡＬＦ－ＡＣＥ对判断Ｓａｒｃ活动性有重要临床意义。     

重组质粒pGEX—6P—1／Sj—FABPc的构建及在大肠杆菌中的表达

目的 构建基因工程菌株、获得重组蛋白Ｓｊ－ＦＡＢＰｃ（日本血吸虫脂肪酸结合蛋白）。方法 用ＰＣＲ法从日本血吸虫ｃＤＮＡ文库中扩增Ｓｊ－ＦＡＢＰｃ基因片段，再将该片段重组于ｐＧＥＭ－Ｔ中并进行ＤＮＡ测序鉴定，经酶切后将目的片段构建成重组质粒ｐＧＥＸ－６Ｐ－１／Ｓｊ－ＦＡＢＰｃ，转化于大肠杆菌ＢＬ２１，ＩＰＴＧ诱导表达。用Ｇｌｕｔａｔｈｉｏｎｅ Ｓｅｐｈａｒｏｓｅ＾ＴＭ ４Ｂ亲和层析柱对表达产物进行纯     

转化生长因子β1 与病毒性肝炎肝纤维化的关系

研究转化生长因子β１（ＴＧＦβ１）与病毒性肝炎肝纤维化的关系。对４５例慢性病毒性肝炎 病毒人肝组织进行了ＴＧＦβ１免疫组织化学检测,同时检测其中２１例血清ＴＧＦβ１及血清透明质酸（ＨＡ）,层粘蛋白（ＬＮ）、Ⅳ型胶原、（Ⅳ－Ｃ）Ⅲ型前原肽（ＰⅢＰ）水平。结果：随着肝纤维化程度加重,ＴＧβ１血清水平上升,肝内表达逐渐明显,二者呈正相关;ＴＧＦβ１的血清水平及肝内表达ＨＡ、ＬＮⅣ－Ｃ、ＰⅢＰ四项肝纤维化     

Idiopathic pulmonary fibrosis. Abnormalities in bronchoalveolar lavage fluid phospholipids

Bronchoalveolar lavage has been used to sample cells and proteins in the distal lung. One of the major secretory products of the alveolar type II epithelial cells, pulmonary surfactant, can be recovered by lavage. Abnormalities in alveolar type II cells are found in biopsies of patients with idiopathic pulmonary fibrosis (IPF), and abnormalities of pulmonary surfactant phospholipids have been reported after diffuse lung injury in animals and in humans. Therefore, we questioned if abnormalities in lavage phospholipids might also occur in IPF, a chronic inflammatory disease of the alveolar epithelium and interstitium, and, if present, would these abnormalities reflect histopathologic changes or predict responsiveness to therapy. Fifteen untreated patients with IPF, diagnosed by open lung biopsy, were studied and were found to have less than half the amount of bronchoalveolar lavage phospholipid as that recovered from healthy volunteers (p less than 0.05). In addition, patients with IPF had a lower proportion of phosphatidylglycerol and a higher proportion of phosphatidylinositol in the recovered phospholipids than did healthy volunteers (p less than 0.05). The severity of these alterations in phospholipid composition correlated with more advanced fibrotic histopathologic changes. Patients with less depression of total phospholipids in lavage improved with corticosteroid therapy, whereas the patients with more severely decreased total phospholipid recovered in lavage did not.     

Proapoptotic Bid is required for pulmonary fibrosis

The molecular mechanisms of pulmonary fibrosis are poorly understood. Previous reports indicate that activation of TGF-beta1 is essential for the development of pulmonary fibrosis. Here, we report that the proapoptotic Bcl-2 family member Bid is required for the development of pulmonary fibrosis after the intratracheal instillation of bleomycin. Mice lacking Bid exhibited significantly less pulmonary fibrosis in response to bleomycin compared with WT mice. The attenuation in pulmonary fibrosis was observed despite similar levels of inflammation, lung injury, and active TGF-beta1 in bronchoalveolar lavage fluid 5 days after the administration of bleomycin in mice lacking Bid and in WT controls. Bleomycin induced similar levels cell death in vitro in alveolar epithelial cells isolated from WT and bid(-/-) mice. By contrast, alveolar epithelial cells from bid(-/-) mice were resistant to TGF-beta1-induced cell death. These results indicate that Bcl-2 family members are critical regulators for the development of pulmonary fibrosis downstream of TGF-beta1 activation.     

Levels of cytokeratin 19 fragments in bronchoalveolar lavage fluid correlate to the intensity of neutrophil and eosinophil-alveolitis in patients with idiopathic pulmonary fibrosis

Cytokeratin 19 (CK19) is a specific cytoskeletal structure for simple epithelia, including bronchial and alveolar epithelial cells (BAEC). Since CK19 is abundant in alveolar epithelial cells, and could be released from injured alveolar epithelium in idiopathic pulmonary fibrosis (IPF), we investigated the levels of CK19 fragments in the bronchoalveolar lavage fluids (BALF) of 16 patients with idiopathic pulmonary fibrosis (IPF) and 12 patients with sarcoidosis using enzyme-linked immunoassay. There were also 19 control subjects (10 asymptomatic smokers and nine non-smokers). BALF from the non-smokers as well as the asymptomatic smokers contained few CK19 fragments (0.2+/-0.2, 1.3+/-0.5 pg ml(-1) respectively). There were significantly high levels of CK19 in the BALF from patients with IPF (7.3+/-1.4 pg/ml; P<0.01 vs. control non-smoker). Even if the levels of CK19 were expressed as relative to the albumin concentration, significantly increased levels of CK19 fragments were noted in BALF from patients with IPF. However, these levels were not found in BALF from patients with sarcoidosis. Importantly, levels of CK19 fragment in BALF were significantly correlated to the number of neutrophils (r = 0.791, P<0.001) and eosinophils (r = 0.771, P<0.001) but not to that of macrophages or lymphocytes in BALF from IPF patients. Our results suggest the usefulness of CK19 measurement in BALF for assessing the presence of bronchiolo-alveolar epithelial injuries in idiopathic pulmonary fibrosis.     

Pathogenesis of pulmonary fibrosis in interstitial lung disease. Alveolar macrophage PDGF(B) gene activation and up-regulation by interferon gamma

Alveolar macrophages are believed to be central in orchestrating the fibrotic response in interstitial lung disease (ILD). To test the hypothesis that macrophages from patients with ILD were dedicated to growth factor production and that this was independent of other indices of macrophage activation, we measured the mRNA of the B chain of PDGF and TGF-beta, as well as HLA-DR-alpha in alveolar macrophages from patients with ILD and from normal control subjects. When alveolar macrophages were examined immediately after lavage, cells from patients with ILD had increased PDGF(B) but similar TGF-beta and HLA-DR-alpha mRNA when compared with control subjects. Discoordinate regulation of these genes was observed when alveolar macrophage PDGF(B) mRNA increased while TGF-beta and HLA-DR-alpha mRNA decreased after culture for 24 h. This response was not disease-related as these changes were similar in cells from patients with ILD and from control subjects. Because a lymphocytic alveolitis is present in many cases of ILD, we asked whether interferon gamma (IFN-gamma) modulated the activation of these genes. In both the patients and the control subjects, PDGF(B) and HLA-DR-alpha, but not TGF-beta, mRNA were increased after incubation with IFN-gamma. These results indicate that PDGF(B) mRNA may be increased in alveolar macrophages in ILD and that PDGF(B), TGF-beta, and HLA-DR-alpha are independently regulated genes in alveolar macrophages, but that IFN-gamma increases both PDGF(B) and HLA-DR-alpha mRNA. We speculate that IFN-gamma induced PDGF(B) gene activation may be an important mechanism by which lymphocytes promote pulmonary fibrosis.     

普通型间质性肺炎和非特异性间质性肺炎肺组织中不同细胞因子的表达及其意义

目的　研究转化生长因子 (TGF) β1、碱性成纤维细胞生长因子 (b FGF)、白细胞介素 8(IL 8)、白细胞介素 13(IL 13)、γ干扰素 (IFN γ)在普通型间质性肺炎 (UIP)和非特异性间质性肺炎(NSIP)肺组织中的分布、表达及意义。方法　经胸腔镜或开胸肺活检获取 5例UIP和 8例NSIP患者的肺组织。对照组 5例 ,来自手术切除的远离肺癌原发灶的周边肺组织。用免疫组化法半定量分析细胞因子的分布及表达。结果　TGF β1、IL 8、b FGF主要分布在肺泡上皮细胞、肺泡巨噬细胞、细支气管上皮细胞 ,UIP组表达强于NSIP组和对照组。IL 13主要分布在肺泡上皮细胞、肺泡巨噬细胞、间质单个核细胞 ,UIP、NSIP组表达无明显差异 ,但均强于对照组。IFN γ主要分布在间质单个核细胞 ,NSIP组表达强于UIP组和对照组。UIP组的IL 13/IFN γ比值为 (2 18± 0 76 ) ,NSIP组为(0 95± 0 2 8) ,对照组为 (0 91± 0 16 ) ,3组比较差异均有显著性 (P值均 <0 0 5 ) ,而NSIP组与对照组比较差异无显著性。对照组只有肺泡巨噬细胞表达上述各细胞因子。结论　TGF β1、IL 8、b FGF在UIP和NSIP患者肺组织中表达强度的不同和IL 13/IFN γ的是否平衡可能参与了UIP和NSIP不同的发病过程。     

B7-1, B7-2 and class II MHC molecules in idiopathic pulmonary fibrosis and bronchiolitis obliterans-organizing pneumonia.

Interstitial lung diseases are thought to be associated with the infiltration of activated T-lymphocytes. To induce an effective immune response, antigen-presenting cells have to not only present antigenic peptide with major histocompatibility complex (MHC) molecules to T-lymphocytes but also express B7 molecules. Therefore, the expression of B7-1, B7-2 and class II MHC molecules was investigated in lung tissues from patients with idiopathic pulmonary fibrosis (IPF) and bronchiolitis obliterans-organizing pneumonia (BOOP), and in normal lung parenchyma as a control, using immunohistochemical localization. B7-1 and B7-2 were aberrantly expressed in bronchiolar and alveolar epithelial cells, and class II MHC molecules were also aberrantly expressed in bronchiolar epithelial cells in IPF. B7-1 was aberrantly expressed in bronchiolar epithelial cells in BOOP. There was no significant difference in the expression of these proteins in alveolar macrophages between IPF and control subjects. However, B7-2 and class II MHC molecule expression in alveolar macrophages was decreased in BOOP compared with that in control subjects. Expression of CD28 and CTLA4, receptors for B7 molecules, was detected in infiltrating lymphocytes in lung tissues in IPF and BOOP. It was concluded that bronchiolar and alveolar epithelial cells may actively participate in the pathophysiology of idiopathic pulmonary fibrosis through the aberrant expression of B7 and class II major histocompatibility complex molecules. The dysregulation of these molecules in epithelial cells may lead to the activation of autoreactive T-lymphocytes, which might contribute to the pathogenesis of fibrosing lung diseases.     

Increased procollagen III aminoterminal peptide-related antigens and fibroblast growth signals in the lungs of patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder characterized by an increased density of inflammatory cells, fibroblasts, and collagen within the lung parenchyma. To gain insights into the mechanisms leading to the increased density of fibroblasts and altered collagen metabolism in the IPF lung, bronchoalveolar lavage fluid from normal subjects and patients with IPF or sarcoidosis was analyzed for (1) the presence of antigenic material related to the aminoterminal propeptide domain of type III procollagen, and (2) fibroblast growth-promoting activity in the extracellular milieu of the lower respiratory tract. Whereas bronchoalveolar lavage fluid (BALF) type III procollagen aminoterminal peptide-related antigen levels in 59 patients with sarcoidosis were similar to the levels of control subjects (p greater than 0.10), 31 patients with IPF had markedly increased levels (12-fold over controls; p less than 0.025, IPF versus controls; p less than 0.01, IPF versus sarcoidosis). Type III procollagen aminoterminal peptide-related antigen levels correlated with an increase in the ability of BALF to stimulate fibroblast proliferation (p less than 0.05). Furthermore, BALF from patients with IPF markedly stimulated human lung fibroblast proliferation in vitro (199% increase, p less than 0.01), whereas lavage fluid from patients with sarcoidosis and from control subjects did not. The enhanced fibroblast proliferation induced by IPF BALF occurred in the absence of serum and exogenous growth factors, suggesting that both competence- and progression-type growth factors were present in the lavage fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

探讨TGFβ1、PDGF、VEGF对特发性肺纤维化诊断及病情评估的临床意义

目的探讨分泌转化生长因子-β、血小板衍生生长因子、血管内皮生长因子在特发性肺纤维化(IPF)患者支气管肺泡灌洗液(BALF)和血清中的表达水平及评估病情进展的临床意义。 方法选择2014年1月至2018年12月在我院呼吸科诊治的35例IPF患者作为观察组,18例肺结节病患者(Ⅰ期)作为对照组;采用免疫印迹观察TGFβ1、PDGF、VEGF在IPF患者血清中是否存在表达;用酶联免疫吸附法(ELISA)观察2组患者BALF和血清中TGFβ1、PDGF、VEGF的表达水平;最后分析IPF患者BALF和血清中TGFβ1、PDGF、VEGF表达水平与肺功能及血氧饱和度的相关性。 结果TGFβ1、PDGF、VEGF细胞印在IPF患者血清存在表达;与对照组相比,BALF及血清中的TGFβ1、PDGF、VEGF表达上调(P<0.05);IPF组患者BALF及血清中TGFβ1、PDGF、VEGF表达水平与肺功能中FVC%、FEV₁%和DLCO%呈负性相关(P<0.05);与血氧饱和度也呈显著负相关(P<0.05)。 结论IPF患者BALF和血清中TGFβ1、PDGF、VEGF的表达水平明显升高;TGFβ1、PDGF、VEGF与患者的肺功能及血氧饱和度呈负相关,可作为评估IPF患者病情的临床评价指标。