Tgf-beta <sub> 1 </ Sub> / Smads In Signal Transduction Pathways In The Lung Muscle Fibroblast Differentiation And Ifn-gamma, The Effect Of Dexamethasone,

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:...