TGF-β/Smad信号通路中关键基因在肺纤维化进程中的功能  

Functions of key genes involved in TGF-β/Smad signaling pathway in progression of pulmonary fibrosis

作　　者：杨慧楠 吕达 王乐 刘春城 姜志艳 赵宏宇[1,2] 蔡禄 YANG Huinan;LYU Da;WANG Le;LIU Chuncheng;JIANG Zhiyan;ZHAO Hongyu;CAI Lu(School of Life Science and Technology,Inner Mongolia University of Science and Technology,Baotou,Inner Mongolia 014010,China;Inner Mongolia Autonomous Region Key Laboratory of Functional Genome Bioinformatics,Inner Mongolia University of Science and Technology,Baotou,Inner Mongolia 014010,China)

机构地区：[1]内蒙古科技大学生命科学与技术学院,内蒙古包头014010 [2]内蒙古科技大学内蒙古自治区功能基因组生物信息学重点实验室,内蒙古包头014010

出　　处：《环境与职业医学》2022年第7期745-751,共7页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金(62071259,31760247);内蒙古自然科学基金(2021MS03007,2019MS08175)。

摘　　要：[背景]转化生长因子-β(TGF-β)/Smad信号通路是调控肺纤维化发生发展的一条重要信号通路,对其调控分子机制的理解仍然有限。[目的]探究纤维化标志物以及TGF-β/Smad信号通路中相关基因在肺纤维化进程中的变化及功能。[方法]建立TGF-β1诱导NIH-3T3成纤维细胞模型,实验分为对照组、TGF-β1处理组。对照组采用生理盐水代替TGF-β1进行同样的培养和处理,TGF-β1处理组用10 ng·mL^(−1) TGF-β1诱导12 h后收集细胞。提取两组细胞RNA,进行转录组测序,经生物信息学分析,筛选获得TGF-β通路中Dcn、Smad3、Smad7、Fbn1、Thbs1、TGF-β1、TGF-β3七种关键基因。采用实时荧光定量PCR法检测I型胶原α1(Collagen1α1)、I型胶原α2(Collagen1α2)、α-平滑肌肌动蛋白(α-SMA)、TGF-β1、TGF-β3五种标志基因和七种通路关键基因mRNA的表达情况。提取两组细胞蛋白,采用Western blotting检测Smad3、磷酸化的Smad3(P-Smad3)、α-SMA三种重要标志蛋白的表达。随后,选取30只SPF级C57 BL/6健康雄性小鼠,随机分成对照组、SiO_(2)染尘28 d组和SiO_(2)染尘56 d组,每组10只。染尘组小鼠每天在SiO_(2)粉尘环境中暴露4 h,隔2 h取出呼吸10 min新鲜空气,分别染尘28、56 d。在两个时间点各处死10只小鼠取肺组织,通过Masson染色检测肺组织纤维化程度变化,提取RNA和蛋白检测上述关键基因和蛋白的表达。[结果]与对照组相比:TGF-β1诱导的NIH-3T3成纤维细胞中,Collagen1α1、Collagen1α2、α-SMA、TGF-β1、TGF-β3五种标志物的基因表达水平均升高(P<0.01);P-Smad3和α-SMA蛋白的表达水平均增加(P<0.01);Dcn和Smad3表达下调(P<0.01),Smad7、Fbn1、Thbs1、TGF-β1、TGF-β3表达上调(P<0.01),与转录组测序的基因表达量变化趋势一致。染尘小鼠肺组织的Masson染色结果显示,随着染尘时间增加,SiO_(2)染尘组小鼠肺组织内的胶原纤维含量也随之增加。与对照组相比,SiO_(2)染尘组小鼠肺组织内�[Background]Although transforming growth factor-β(TGF-β)/Smad signaling pathway is important in regulating the occurrence and development of pulmonary fibrosis,the pathogenesis of pulmonary fibrosis remains elusive.[Objective]To explore the functions of genes associated with TGF-β/Smad signaling pathway in the progression of pulmonary fibrosis.[Methods]A NIH-3T3 fibroblast model induced by TGF-β1 was established.The experiment samples were divided into a control group and a TGF-β1 treatment group.The control group was exposed to normal saline,while the TGF-β1 treatment group was exposed to 10 ng·mL^(−1) TGF-β1 for 12 h.The RNAs of the two groups were extracted,sequenced,and analyzed by bioinformatics methods to identify seven key genes in TGF-βpathway,including Dcn,Smad3,Smad7,Fbn1,Thbs1,TGF-β1,and TGF-β3.The gene expression levels of five markers[Collagen1α1,Collagen1α2,α-smooth muscle actin(α-SMA),TGF-β1,and TGF-β3]and the seven key genes were detected by quantitative real-time PCR(qRT-PCR).The proteins of the two groups were extracted.The important marker protein expression levels of Smad3,the phosphorylation of Smad3(P-Smad3),andα-SMA were detected by Western blotting.At the same time,30 healthy SPF-grade C57BL/6 mice were randomly divided into three groups,with 10 mice in each group:a control group,a SiO_(2) inhalation exposure group for 28 d(10 mice),and a SiO_(2) inhalation exposure group for 56 d(10 mice).The mice in the two treatment groups were exposed to a natural SiO_(2) environment for 4 h per day with a 10-min pause for breathing fresh air at 2 h intervals.The lung tissues of the mice were taken after execution.The changes of pulmonary fibrosis were detected by Masson staining,and mRNAs and proteins were extracted to detect the expression of the above key genes and proteins.[Results]The expression levels of the five marker genes Collagen1α1,Collagen1α2,α-SMA,TGF-β1,and TGF-β3 were significantly increased in the TGF-β1-induced NIH-3T3 fibroblasts than those in the control gr

关 键 词：肺纤维化 转化生长因-β TGF-Β/SMAD信号通路 NIH-3 T3细胞 转录组测序 

分 类 号：R114[医药卫生—卫生毒理学]