【摘要】 目的:探讨从博来霉素致肺纤维化小鼠肺组织中筛选出的高迁移率族蛋白B1(HMGB1)亚家族成员HMGB34367,作为转录因子对α-平滑肌肌动蛋白(α-SMA)基因表达的影响。方法:①克隆与构建HMGB34367真核表达载体及α-SMA启动子红色荧光报告质粒。②构建质粒共转染气道上皮细胞系16HBE,荧光显微镜观察重组转化生长因子-β1(rhTGF-β1)刺激下红色荧光的表达。③电泳迁移率(EMSA)及超迁移实验分析HMGB34367和α-SMA启动子CArGB基序的特异性结合活性。④RT-PCR检测细胞转染HMGB34367真核表达载体HMGB34367-pcDNA3.0对内源性α-SMA基因表达的影响。结果:细胞过表达HMGB34367能活化α-SMA启动子,转染HMGB34367-pcDNA3.0质粒的16HBE细胞核蛋白,显著增强了与α-SMA启动子CArGB序列特异结合,过表达HMGB34367诱导了α-SMA基因表达。结论:HMGB34367基因编码蛋白能作为转录因子特异性地诱导α-SMA启动子的转录激活,上调αS-MA基因表达。提示致纤维化环境因子通过诱导HMGB34367表达使αS-MA基因转录激活,可能在气道重塑和肺纤维化的病理过程中发挥重要作用。更多还原

【Abstract】 Objective: To demonstrate the influence of HMGB34367,a subfamily member of the high-mobility group protein B1(HMGB1),as a transcriptional factor of the α-SMA gene expression.Methods: We cloned HMGB34367 cDNA from the fibrotic lung tissue of the Balb/C mouse treated with BLM by RT-PCR,sub-cloned it into a eukaryotic expression vector pcDNA 3.0 or pEGF-N2,and constructed a report plasmid,pDsRed-SMA,encoding red fluorescence(RFP) driven by α-SMA promoter.Following the co-transfection of pDsRed-SMA and HMGB34367-pcDNA3.0/ pEGF-N2-HMGB34367 in the cultured 16HBE cells,we tested the expression of the RFP in the absence and presence of TGFβ1 by fluorescence microscopy.After the transfection of HMGB34367-pcDNA3.0,the nucleus extracts from the transfected cells were subjected to electrophoretic mobility shift assay(EMSA) for the detection of the binding activity of HMGB34367 with α-SMA promoter CarGB motif.RT-PCR was performed for the evaluation of the α-SMA gene expression in the cells.Results: The over expression of HMGB34367 activated the α-SMA promoter and enhances the expression of the α-SMA gene.An increased binding activity of HMGB34367 with CarGB motif was detected by EMSA in the transfected cells.Conclusion:HMGB34367,a member of HMGB1 family,could act as a transcription factor for the transcriptional activation of the α-SMA gene,which may play an important role in the development of lung fibrosis.更多还原