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
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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、SiO2染尘28 d组和SiO2染尘56 d组，每组10只。染尘组小鼠每天在SiO2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，SiO2染尘组小鼠肺组织内五种标志基因的表达水平均上调（P < 0.01）；Smad3蛋白表达无明显变化，P-Smad3和α-SMA表达水平较对照组明显升高（ P < 0.01）； Dcn、Smad3表达下调（P < 0.01）， Smad7、Fbn1、Thbs1、TGF-β1、TGF-β3表达上调（P < 0.01） ，且都呈现随染尘天数的增加而下降或升高的趋势。上述五种标志基因、三种重要标志蛋白以及七种通路关键基因的表达均与TGF-β1诱导的NIH-3T3成纤维细胞中的表达趋势一致。
结论 TGF-β1诱导成纤维细胞中肺纤维化相关标志基因和蛋白表达水平变化有统计学意义，染尘小鼠肺纤维化特征明显。Dcn、Smad3、Smad7、Fbn1、Thbs1、TGF-β1、TGF-β3七种基因可能参与了TGF-β/Smad信号通路对肺纤维化的调控。
Abstract: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-β pathway, including Dcn,Smad3,Smad7,Fbn1,Thbs1,TGF-β1, andTGF-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), and α-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 SiO2 inhalation exposure group for 28 d (10 mice), and a SiO2 inhalation exposure group for 56 d (10 mice). The mice in the two treatment groups were exposed to a natural SiO2 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 genesCollagen1α1, Collagen1α2, α-SMA,TGF-β1, andTGF-β3 were significantly increased in the TGF-β1-induced NIH-3T3 fibroblasts than those in the control group (P < 0.01); the expression levels of P-Smad3 and α-SMA proteins increased significantly ( P < 0.01); the expression results of the seven key genes screened in the TGF pathway were that Dcn and Smad3 were obviously down-regulated (P < 0.01), and Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 were obviously up-regulated (P < 0.01). The changes in gene expression levels of the transcriptome sequencing showed the same trend. The results of Masson staining showed that the content of collagen fibers in the lung tissues also increased in the SiO 2 inhalation exposure groups over time. In the mouse experiment, five marker genes were obviously up-regulated compared with the control group (P < 0.01); no obvious change was found in the expression of Smad3 protein, and the expression levels of P-Smad3 and α-SMA were obviously higher in the SiO 2 exposure groups than those in the control group (P < 0.01); the expression levels of Dcn and Smad3 showed a down-regulated trend, while the expression levels of Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 showed an up-regulated trend with the increase of SiO2 inhalation exposure days (P < 0.01). The expression levels of the above five marker genes, three important marker proteins, and seven key genes were consistent with the expression trends of TGF-β1-induced NIH-3T3 fibroblasts.
Conclusion The expression levels of pulmonary fibrosis-related marker genes and proteins change significantly in TGF-β1-induced fibroblast cells, and the lung tissues of mice under natural SiO2 inhalation exposure has obvious fibrosis characteristics. Seven genes (Dcn,Smad3, Smad7, Fbn1,Thbs1, TGF-β1, and TGF-β3) may be involved in the regulation of pulmonary fibrosis by the TGF-β/Smad signaling pathway.
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