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Follistatin-like 1是肺纤维化的重要调节因子

作　者: 董莺莺
导　师: 宁文
学　校: 南京大学
专　业: 生物学
关键词: 特发性肺纤维化(IPF) 博莱霉素 FStl1 细胞外基质肌成纤维细胞上皮细胞间质转型(EMT) TGF-β1
分类号: R563
类　型: 博士论文
年　份: 2011年
下　载: 13次
引　用: 0次
阅　读: 论文下载

内容摘要

特发性肺纤维化(IPF)是一种病因不明的,高度致死的慢性渐进性肺疾病,以成纤维细胞／肌纤维母细胞聚积和细胞外基质(ECM)过度沉积为特征。Fstl1是TGF-β1可诱导产生的分泌型胞外糖蛋白,最近发表的microarray数据表明Fstll在IPF患者的肺组织中表达量较正常人升高。在本研究中,我们发现Fstl1在IPF患者的肺组织和原代成纤维细胞中的表达量高于正常人。并且,Fstl1在博莱霉素所诱导的肺纤维化模型中也被诱导表达。因此,我们推测Fstl1在IPF病理过程中起到一定的作用。为了验证这一假设,Fstl1+/-和野生型小鼠经气管注射博莱霉素诱导形成肺纤维化。相对于野生型小鼠,Fstl1+/-小鼠具有正常的博莱霉素所引起的急性免疫反应。但是死亡曲线,组织学分析,羟脯氨酸测定以及免疫印迹分析都表明单倍缺失Fstl1明显抑制博莱霉素诱导形成的纤维化过程。ECM蛋白的过度沉积和重塑最终导致肺纤维化和肺泡功能的丧失。在肺组织和原代成纤维细胞中,单倍缺失Fstl1明显降低了博莱霉素所诱导的ECM蛋白的表达。另外,作为能够诱导ECM形成的TGF-β1在小鼠肺成纤维细胞中能够诱导Fstll的表达。我们发现,来源于Fstl1+/-或Fstl1+/-小鼠的肺成纤维细胞在TGF-β1处理后, ECM蛋白的表达明显低于正常细胞,这表明TGF-βl诱导产生ECM蛋白的表达是依赖于Fstll的。作为IPF主要的效应细胞,肌纤维母细胞是ECM合成聚积的主要细胞类型。在博莱霉素诱导肺纤维化的小鼠模型中,我们发现Fstl1+/-小鼠的肺组织中α-SMA的表达量明显低于正常小鼠,这表明在纤维化过程中内源的Fstl1调节了肌纤维母细胞的聚积。TGF-β1通过诱导α-SMA的表达来促进成纤维细胞的活化。在小鼠原代肺成纤维细胞中,缺失Fstl1抑制了TGF-β1所诱导的α-SMA的表达,进一步表明Fstl1在博莱霉素所诱导的成纤维细胞活化过程中起作用。现在认为肺上皮细胞可以通过上皮细胞间质转型(EMT)形成肌纤维母细胞,而EMT这一过程通常由TGF-β所介导。α-SMA和Pro-SPC的双染实验表明单倍敲除Fstl1明显减少了体内的EMT过程。利用A549细胞进行的体外实验进一步发现,过表达FStl1通过Smad和MAPK通路促进TGF-β1所诱导形成的EMT过程,Smad2/3,ERK或JNK的化学抑制剂明显抑制了Fstl1在TGF-β1所诱导的EMT过程中的作用。最后,我们第一次证明了Fstlll在体外能够直接与TGF-β1或TGF-β二型受体结合,但不与TGF-β一型受体结合。总之,单倍敲除Fstl1通过减少ECM沉积和EMT过程明显地抑制了博莱霉素所诱导的肺纤维化,但并不影响急性炎症反应。Fstl1通过与TGF-β受体复合物作用来调节下游的Smad和MAPK信号,来进一步调节TGF-β1所诱导的EMT过程。我们的研究揭示了Fstll在博莱霉素诱导的肺纤维化发生过程中的重要作用,并为肺纤维化的研究及治疗提供了新的靶向目标。

全文目录

Abstract  7-9
中文摘要  9-11
Abbreviations  11-12
Chapter1:Literature review of idiopathic pulmonary fibrosis  12-33
  1.1 Clinical description of IPF  12-14
  1.2 Pathogenesis of IPF  14-21
    1.2.1 IPF results from epithelial injury and abnormal wound healing  14-16
    1.2.2 Myofibroblasts in IPF  16
    1.2.3 Origin of myofibroblasts in IPF  16-19
    1.2.4 TGF-β1:a novel target for treatment of IPF  19-21
  1.3 Animal models of pulmonary fibrosis  21-24
  References  24-33
Chapter 2:Hyploinsufficiency of Fstl1 suppresses bleomycin-inducedpulmonary fibrosis  33-96
  1. Introduction  33-34
  2. Materials and methods  34-45
  3. Results  45-85
    Section Ⅰ. Altered expression of FSTL1 in IPF patients  45-51
      1.1 Microarray analysis of FSTL1 in IPF patients and controls  45-47
      1.2 FSTL1 expression in lung tissues of IPF patients and controls  47-48
      1.3 FSTL1 expression in PPFs from IPF patients and controls  48-49
      1.4 Fstl1 expression in lung tissues of mice after bleomycin treatment  49-50
      1.5 Fstl1 expression PPFs from mice after bleomycin treatment  50
      1.6 Localization of Fstl1 in lungs from mice after bleomycin treatment  50-51
      1.7 Conclusions  51
    Section Ⅱ.Fstl1~(+/-) mice have an attenuated fibrotic phenotype afterbleomycin-induced lung injury  51-60
      2.1 Expression of Fstll in Fstl1~(+/-) mice and WT controls after bleomycintreatment  51-53
      2.2 Survival curve of Fstl1~(+/-) mice and WT controls after bleomycin treatment  53-54
      2.3 Histological analysis of lung tissues from Fstl1~(+/-) mice and WT controls afterbleomycin treatment  54-56
      2.4 Collagen deposition of lung tissues from Fstl1~(+/-) mice and WT controls afterbleomycin treatment  56-57
      2.5 ECM deposition of lung tissues from Fstl1~(+/-) mice and WT controls afterbleomycin treatment  57-59
      2.6 Conclusions  59-60
    Section Ⅲ.Fstl1~(+/-) mice showed a normal acute response to bleomycin-inducedlung injury  60-64
      3.1 Total and differential inflammatory cells in BAL fluid  60-61
      3.2 Leukocyte and macrophage Subsets in whole lung tissues  61-63
      3.3 Th1/2 cytokines in BAL fluid and lung tissues  63
      3.4 Conclusions  63-64
    Section Ⅳ Fstl1~(+/-) mice exhibited reduced EMT process in response to bleomycin  64-69
      4.1 Decreased accumulation of myofibroblasts in Fstl1~(+/-) lungs afterbleomycin-induced injury  64-66
      4.2 Reduced EMT in Fstl1~(+/-) lungs during tissue injury  66-68
      4.3 Conclusions  68-69
    Section Ⅴ.Fstl1 exerts its pro-finbrotic role through modulating TGF-β signaling  69-85
      5.1 TGF-β1 induced Fstl1 expression in mouse lung fibroblasts and epithelial cells  69-70
      5.2 Fstl1 deletion suppresses TGF-β1-induced myofibroblasts activation and ECMproduction in lung fibroblasts  70-72
      5.3 Fstl1 promotes TGF-β1-induced EMT in vitro  72-75
      5.4 Fstl1 modulates TGF-β1-induced EMT via Smad signaling  75-79
      5.5 Fstl1 modulates TGF-β1-induced EMT via MAPK signaling  79-81
      5.6 Fstl1 binds to TGF-β1 in vitro  81-84
      5.7 Conclusions  84-85
  4. Conclusions and Discussions  85-90
  References  90-96
Acknowledgments  96-97
Publication  97-98

Follistatin-like 1是肺纤维化的重要调节因子

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