TGF-β_1/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
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摘要
一、Smads蛋白在TGF-β_1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-β_1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-β_1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-β_1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pCS_2-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pCS_2-Smad2质粒片段形成pCS_2-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-β_1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-β_1干预后通过Western blot分析α-SMA基因的蛋白表达。
结果：
1、5ng／ml TGF-β_1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-β_1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-β_1对α-SMA基因启动子活性及蛋白表达的
I: The Role of Smads Signaling during Lung Fibroblasts-Myofibroblasts
Differentiation Induced by Transforming Growth Factor-β_1 in vitro
Objective: Myofibroblasts play an important role in the fibrotic pathogenesis.The
expression of alpha-smooth muscle actin (α -SMA) is a marker of differentiation of
myofibroblasts. A family of cytoplasmic proteins called Smads is found to mediate
intracellular signaling of TGF-β_1. So, the present study was undertaken to further
explore the role of Smads signaling in the regulation of α -SMA gene expression
during lung fibroblasts-myofibroblasts differentiation induced by TGF-β_1.
Methods:
1、 Human fetal lung fibroblasts(HFL-F) were cultured in vitro. After induced by
TGF-β_1, Western blot, RT-PCR was used to analyses α -SMA gene protein and
mRNA expression. And cell morphological transformation was investigated by
immunofluorescent technique.
2、 Construction of luciferase reporter plasmid containing human a-SMA gene
promote (p895-Luc) and mutant Smad2 plasmid (pSmad2~(mut)): The human a-SMA
gene promoter (-895~+9bp) was cloned by PCR from human genomic DNA and
was inserted into vector pGa(?)3-basic at MluI-XhoI site and form p895-Luc. Smad2~(mut)
were generated by PCR-based mutagenesis from pCS_2-Smad2 plasmid DNA and PCR
products were inserted into the Bgl II -Xho I sites of pSmad2 plasmid. Other plasmids
including pSmad2, pSmad3, pSmad7 and pSmad3~(mut) were provided kindly.
3, p895-Luc was performed transient co-transfection with different Smad expression
plasmids by using Fugene 6.0 reagent in HFL-F, respectively. The lucifease and
β-galactosidase activities were measured after treated by TGF-β_1.
4, The different Smad expression plasmids were performed transient transfection by
using Fugene 6.0 reagent in HFL-F. The α -SMA protein expression was analyses by
Western blot after treated by TGF-β_1.
Results:
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-β_1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-β_1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-β_1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pCS_2-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pCS_2-Smad2质粒片段形成pCS_2-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-β_1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-β_1干预后通过Western blot分析α-SMA基因的蛋白表达。
结果：
1、5ng／ml TGF-β_1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-β_1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-β_1对α-SMA基因启动子活性及蛋白表达的
I: The Role of Smads Signaling during Lung Fibroblasts-Myofibroblasts
Differentiation Induced by Transforming Growth Factor-β_1 in vitro
Objective: Myofibroblasts play an important role in the fibrotic pathogenesis.The
expression of alpha-smooth muscle actin (α -SMA) is a marker of differentiation of
myofibroblasts. A family of cytoplasmic proteins called Smads is found to mediate
intracellular signaling of TGF-β_1. So, the present study was undertaken to further
explore the role of Smads signaling in the regulation of α -SMA gene expression
during lung fibroblasts-myofibroblasts differentiation induced by TGF-β_1.
Methods:
1、 Human fetal lung fibroblasts(HFL-F) were cultured in vitro. After induced by
TGF-β_1, Western blot, RT-PCR was used to analyses α -SMA gene protein and
mRNA expression. And cell morphological transformation was investigated by
immunofluorescent technique.
2、 Construction of luciferase reporter plasmid containing human a-SMA gene
promote (p895-Luc) and mutant Smad2 plasmid (pSmad2~(mut)): The human a-SMA
gene promoter (-895~+9bp) was cloned by PCR from human genomic DNA and
was inserted into vector pGa(?)3-basic at MluI-XhoI site and form p895-Luc. Smad2~(mut)
were generated by PCR-based mutagenesis from pCS_2-Smad2 plasmid DNA and PCR
products were inserted into the Bgl II -Xho I sites of pSmad2 plasmid. Other plasmids
including pSmad2, pSmad3, pSmad7 and pSmad3~(mut) were provided kindly.
3, p895-Luc was performed transient co-transfection with different Smad expression
plasmids by using Fugene 6.0 reagent in HFL-F, respectively. The lucifease and
β-galactosidase activities were measured after treated by TGF-β_1.
4, The different Smad expression plasmids were performed transient transfection by
using Fugene 6.0 reagent in HFL-F. The α -SMA protein expression was analyses by
Western blot after treated by TGF-β_1.
Results:
引文
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