Fstll对于出生时肺的成熟起至关重要的作用

论文摘要

肺的正常发育和功能的形成对于胎儿出生时呼吸能否很快适应外界空气的过程中至关重要。在妊娠后期,胎肺要经历了一个重要的成熟过程,包括肺泡Ⅰ型和Ⅱ型上皮细胞的分化和肺泡表面活性剂的生成。这一进程若受到阻碍会导致新生儿呼吸窘迫综合症。我们发现,含有Follistatin-module的分泌糖蛋白Fstl1,对于肺成熟和维持肺表面活性蛋白稳态至关重要。Fstl1在肺发育过程中广泛表达并在胚胎期E17.5表达量最高。Fstl1基因敲除小鼠因肺部发育受损,出生后不久即死于呼吸衰竭。虽然和胚胎期18.5天的野生型的胎肺相比,Fstl1基因敲除小鼠有着类似大小,叶数,肺干重/体重比,但它的肺扩张不全。组织学分析显示其间质明显增厚,肺泡塌陷。进一步的研究表明,Fstl1缺失并不影响假腺体期肺分支形态的发生,但从胚胎期第16.5起推迟了囊泡化的发育。在假腺体期Fstl1基因敲除小鼠的上皮细胞增殖率增加可能是导致其肺囊泡壁异常增厚原因。通过透射电镜发现,在胚胎期第18.5天,Fstl1基因敲除小鼠的肺中不成熟的Ⅱ型上皮细胞数目显著增加。在野生型胎肺中清晰可见对气体交换至关重要的由扁平肺泡Ⅰ型上皮细胞和血管内皮细胞共同构成的气血屏障。相比之下,Fstl1基因敲除小鼠的囊泡壁由于不成熟的Ⅱ型上皮细胞数目增多而肺泡Ⅰ型上皮细胞和血管内皮细胞数目减少致使气血屏障增厚。我们还通过实时定量RT-PCR和免疫组化检测了T1α和Aqp5的表达（肺泡Ⅰ型上皮细胞的标记物）和Sftpc（肺泡Ⅱ型上皮细胞的标记物）的表达。Sftpc RNA表达水平升高,而Tla及Aqp5的表达严重降低。免疫印迹分析表明,Fstl1缺失并没有显着影响肺泡表面活性蛋白SP-B和SP-C前体蛋白的表达。然而,SP-B和SP-C蛋白的成熟体在Fstl1基因敲除小鼠的肺中急剧下降。因此,不成熟的Ⅱ型上皮细胞不能分泌足够的成熟肺泡表面活性蛋白致使囊泡功能异常,从而导致Fstl1基因敲除小鼠肺不张。总而言之,这些缺陷有可能是导致纯合子小鼠出生致死的原因。通过微阵列分析,我们发现在胚胎期18.5的基因敲除小鼠的肺中许多信号通路受到影响,如调节脂类稳态,细胞粘连胞外基质重塑,免疫反应的信号通路等等。此外,Fstl1基因的缺失导致调控肺上皮细胞增殖和分化的生长因子CTGF下调。在分子水平上,Fstl1可直接与BMP4相结合,负调控BMP4/Smad1/5/8信号通路,并能抑制BMP4诱导下游基因的表达。通过Noggin或Dorsomorphin降低BMP信号活性可以挽救Fstl1缺陷小鼠肺不张的表型。因此,体内和体外的证据表明Fstl1部分通过BMP4信号通路调节肺的发育和肺泡的成熟。我们也使用人表面活性蛋白C（SPC）启动子特异性激活Fstl1基因在小鼠肺上皮细胞中持续表达（SPC-Fstl1转基因小鼠）。实时定量RT-PCR和免疫印迹证实在转基因小鼠的肺中Fstl1表达量显著升高。免疫组织化学分析表明,早在胚胎期12.5天,Fstl1已在肺上皮细胞中广泛激活。然而,过表达Fstl1的小鼠肺形态发生和肺功能并没有改变。我们还进一步的将转基因小鼠和Fstl1敲除杂合子小鼠交配得到复合小鼠（SPC-Fstl1;Fstl1+/-）。Fstl1在肺上皮细胞过表达不能挽救Fstl1基因敲除小鼠肺不张的表型,不会改变其肺部结构和产后的生存。

论文目录

• Abstract
• 中文摘要
• Abbreviations
• Review:Lung development and Fstl1
• 1.1 Lung development
• 1.1.1 Overview of Lung development
• 1.1.2 Lung branching morphogenesis
• 1.1.3 Lung maturation and differentiation
• 1.1.4 Differentiation of the Epithelial Cells of the lung
• 1.1.5 Epithelial Differentiation in the Mouse Trachea
• 1.1.6 Mesenchymal cell differentiation
• 1.2 Biology of Fstl1
• 1.2.1 Structure of Fstl1
• 1.2.2 Family members of Fstl1
• 1.2.3 Distribution of Fstl1
• 1.2.4 Fstl1 and rheumatoid arthritis
• 1.2.5 Fstl1 and cell proliferation,migration and differentiation
• References
• Chapter 1:Expression patterns of Fstl1 during lung development
• Introduction
• Materials and methods
• Results
• 1.1 Expression patterns of Fstl1 during lung development by in situ hybridization, real-time PCR and western blotting analysis
• 1.2 Comparison of mRNA expression levels for Fstl1,Follistatin and Noggin during mouse lung development
• 1.3 Expression of Fstl1 in isolated primary lung cells
• Discussions
• References
• Chapter 2:Phenotypical analysis of pulmonary system in Fstl1
• deficient mice
• Introduction
• Materials and methods
• Results
• 2.1 Impaired lung morphogenesis in Fstl1 deficient mice
• 2.2 Lung branching morphogenesis and Cell proliferation
• 2.3 Impaired lung epithelium differentiation in Fstl1 deficient mice
• -/- mice'>2.4 Surfactant dysfunction of immature AEC2 cells in Fstl1^-/-mice
• 2.5 Fstll in differentiation of Alveolar Epithelial Cells
• 2.6 Deletion of Fstl1 leads to abnormal trachea epithelium differentiation
• -/- mice lung'>2.7 ECM change in E18.5 Fstl1^-/- mice lung
• Discussions
• References
• Chapter 3:Fstl1 regulates AECs differentiation via BMP4/Smad signaling
• Introduction
• Materials and methods
• Results
• 3.1 Fstl1 binds to BMP4 and negatively regulates Smad signaling in vitro
• -/- #89 mice'>3.2 Reducing BMP signaling activity rescues pulmonary atelectasis of Fstl1^-/- #89 mice
• Discussions
• References
• Chapter 4:Microarray analysis of Fstl1 deficient mice
• Introduction
• Materials and methods
• Results
• -/- mice:microarray #101 analyses'>4.1 Genes differentially expressed between WT and Fstl1^-/- mice:microarray #101 analyses
• 4.2 Validation of gene expression differences found by microarray data
• Discussions
• References
• Chapter 5:Ectopic expression of Fstl1 in lung epithelial cells
• Introduction
• Materials and methods
• Results
• 5.1 Generation of SPC-Fstl1 transgenic mice
• 5.2 Phenotypical analysis of SPC-Fstl1 mice
• -/- mice'>5.3 Over expression of Fstl1 in lung epithelial cells doesnot rescue the atelectasis #117 phenotype of Fstl1^-/-mice
• 5.4 Over expression of Fstl1 in lung epithelial cells in Bleomycin induced #118 pulmonary fibrosis
• Discussions
• References
• Acknowledgments
• Publication