转化生长因子β1与Smad3信号传导在肺移植术后闭塞性细支气管炎中的作用  

The role of transforming growth factor-β1/Smad3 signaling in bronchiolitis obliterans following lung transplantation

作　　者：刘宏旭[1] 李玉[1] 赵成海[2] 刘洋[3] 张其刚[1] 丛巍[1] 兰心刚[1] 许顺[1] 韩立波[1] 张林[1] 

机构地区：[1]中国医科大学附属第一医院胸外科,沈阳110001 [2]中国医科大学病理生理教研室 [3]中国医科大学病理教研室

出　　处：《中华医学杂志》2007年第29期2069-2073,共5页National Medical Journal of China

摘　　要：目的探讨肺移植术后闭塞性细支气管炎的发生机理及转化生长因子(TGF)β1/Smad3信号传导途径在此病理过程中的意义。方法闭塞性细支气管炎动物模型采用 Smad3野生型和基因敲除小鼠进行的同种异体气管异位移植,免疫组织化学检测肌纤维母细胞分化的标志物——α平滑肌肌动蛋白(αSMA)的存在及差异。采用原代培养 Smad3野生型和敲除型气管纤维母细胞,经 TGF-β1处理,通过免疫荧光、Western 印迹和 DNA 凝胶电泳迁移率检测手段,检测 Smad3蛋白的激活。并用免疫荧光染色检测细胞骨架聚合能力和αSMA 分子的不同表达;Western 印迹和 RT-PCR法检测αSMA 在转录和蛋白水平的差异。结果在发生闭塞性细支气管炎的受累气道中,发现有肌纤维母细胞的存在,且其在 Smad3野生型中的数量明显多于敲除型(t=2.125,P=0.040)。对纤维母细胞进行的离体研究发现,TGF-β1诱导 Smad3激活,表现为蛋白磷酸化、细胞核转位和 DNA 结合增加。在 Smad3野生型的纤维母细胞,TGF-β1引起细胞骨架聚合能力增强、αSMA 在转录和蛋白水平的表达增强,即向肌纤维母细胞的分化增加;而在缺乏 Smad3的纤维母细胞中,TGF-β1诱导的肌纤维母细胞分化明显减少(t=2.080,P=0.027;t=1.982,P=0.032)。结论 TGF-β1主要通过激活Smad3依赖性信号传导途径,来促使纤维母细胞向肌纤维母细胞的转化和αSMA 蛋白的增加,最终导致闭塞性细支气管炎的发展。Objective To investigate the mechanism underlying bronchiolitis obliterans （OB） following lung transplantation and the significance of transforming growth factor （TGF）-β1/Smad3 signal pathway in this pathological process. Methods The tracheas of BALB/c mice were transplanted into the subcutaneous tissues of a Smad3exS/ex8 gene knock-out Swiss black mouse and a Smad3 wild-type Swiss black mouse. Forty-two days later the tracheas were taken out. Immunocytochemistry was used to detect the a-smooth muscle actin （ctSMA） , a marker of fibroblast-myofibroblast differentiation. The tracheas of Smad3 knock-out and wild type mice were taken out, broken to pieces, and cultured to obtain the fibroblasts. The tracheal fibroblasts in primary culture were treated with TGF-β1. The activation of Smad3 molecules was investigated with immunocytochemistry, Western blotting and DNA elctrophoresis mobility gel shift assay （EMSA）. Immunocytochemistry staining was also employed to detect the cytoskeletal polymerization and αSMA immunofluorescence after incubation with TGF-β1 ; Western blotting and RT-PCR was conducted to detect the difference of αSMA at transcriptional and protein level. Results The number of αSMA positive myofibroblasts was great in the experimental OB models produced be transplantation of heterogeneous trachea from Smad3 wild type mice and was very small in the OB model produced be transplantation of heterogeneous trachea from Smad3 knock-out mice （t = 2. 125, P=0. 040）. Western blotting showed that in vitro experiment showed that phosphorylation of Smad3 protein was increased in the fibroblasts treated withTGF-β1 and was almost absent in those not treated with TGF-β1. EMSA showed that DNA binding was increased in the fibroblasts treated with TGF-β1 and was almost absent in those not treated with TGF-β1. Immunofluorescence staining showed that the cytoplasm of the fibroblasts not treated withTGF-β1 was Smad3 positive, however, the nuclei of the fibroblasts treated withTGF-β1 was Smad3 p

关 键 词：肺移植 细支气管炎 闭塞性 移植 异位 信号传导 

分 类 号：R655.3[医药卫生—外科学]