TGF-β₁/Smads信号转导途径在肺肌成纤维细胞分化中的作用及IFN-γ、地塞米松的影响

【作者】 谷丽；

【导师】 朱元珏； 许文兵；

【作者基本信息】 中国协和医科大学 ， 内科呼吸病学， 2005， 博士

【摘要】 一、Smads蛋白在TGF-β₁体外诱导肺成纤维细胞向肌成纤维细胞分化中作用 目的：肺肌成纤维细胞在肺纤维化发生机制中起重要作用，其分化标志是α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）的表达。研究发现TGF-β₁可通过Smads蛋白家族成员传递信号发挥生物作用，因此本研究主要探讨在TGF-β₁诱导肺成纤维细胞向肌成纤维细胞分化过程中，各Smad蛋白对α-SMA基因表达的调控作用。 方法： 1、体外培养人胚胎肺成纤维细胞（HFL-F），TGF-β₁干预后通过Western blot及RT-PCR分析α-SMA基因mRNA和蛋白水平表达的变化，同时用免疫荧光法观察细胞形态改变。 2、构建含人α-SMA基因启动子序列的荧光素酶报告基因质粒（p895-Luc）和Smad2突变表达质粒(pSmad2^mut)：以人基因组DNA为模板用PCR扩增人α-SMA基因启动子-895～+9bp序列，插入到pGal3-basic质粒的MluⅠ和XhoⅠ酶切位点之间形成p895-Luc。突变Smad2序列是通过以pCS₂-Smad2质粒为模板用PCR方法获取，然后将其插入到经BglⅡ和XhoⅠ酶切后的pCS₂-Smad2质粒片段形成pCS₂-Smad2^mut。Smad2、Smad3、Smad7和Smad3^mut表达质粒为他人惠赠。 3、用Fugene6.0将p895-Luc分别和各种Smad表达质粒瞬时共转染至HFL-F中，TGF-β₁干预后测定其荧光素酶和β—半乳糖苷酶的活性。 4、用Fugene6.0将各种Smad表达质粒瞬时转染至HFL-F中，TGF-β₁干预后通过Western blot分析α-SMA基因的蛋白表达。 结果： 1、5ng／ml TGF-β₁作用4天能诱导肺成纤维细胞向肌成纤维细胞分化。 2、过表达Smad3能增强TGF-β₁诱导的α-SMA基因启动子活性和其蛋白表达，而过表达Smad3^mut能抑制这种增强作用，但没有使其恢复到基础水平，两者对α-SMA基因的基础表达没有影响。 3、过表达Smad2和Smad2^mut不影响TGF-β₁对α-SMA基因启动子活性及蛋白表达的更多还原

【Abstract】 I: The Role of Smads Signaling during Lung Fibroblasts-MyofibroblastsDifferentiation Induced by Transforming Growth Factor-β₁ in vitroObjective: Myofibroblasts play an important role in the fibrotic pathogenesis.Theexpression of alpha-smooth muscle actin （α -SMA） is a marker of differentiation ofmyofibroblasts. A family of cytoplasmic proteins called Smads is found to mediateintracellular signaling of TGF-β₁. So, the present study was undertaken to furtherexplore the role of Smads signaling in the regulation of α -SMA gene expressionduring lung fibroblasts-myofibroblasts differentiation induced by TGF-β₁.Methods:1、 Human fetal lung fibroblasts（HFL-F） were cultured in vitro. After induced byTGF-β₁, Western blot, RT-PCR was used to analyses α -SMA gene protein andmRNA expression. And cell morphological transformation was investigated byimmunofluorescent technique.2、 Construction of luciferase reporter plasmid containing human a-SMA genepromote （p895-Luc） and mutant Smad2 plasmid (pSmad2^mut): The human a-SMAgene promoter （-895⁺9bp） was cloned by PCR from human genomic DNA andwas inserted into vector pGa（?）3-basic at MluI-XhoI site and form p895-Luc. Smad2^mutwere generated by PCR-based mutagenesis from pCS₂-Smad2 plasmid DNA and PCRproducts were inserted into the Bgl II -Xho I sites of pSmad2 plasmid. Other plasmidsincluding pSmad2, pSmad3, pSmad7 and pSmad3^mut were provided kindly.3, p895-Luc was performed transient co-transfection with different Smad expressionplasmids by using Fugene 6.0 reagent in HFL-F, respectively. The lucifease andβ-galactosidase activities were measured after treated by TGF-β₁.4, The different Smad expression plasmids were performed transient transfection byusing Fugene 6.0 reagent in HFL-F. The α -SMA protein expression was analyses byWestern blot after treated by TGF-β₁.Results:更多还原