α肌动蛋白基因转录激活因子HMGB34367的克隆和调控功能研究  

Identification of a novel transcriptional factor HMGB34367 for activation of α-smooth muscle actin gene in relation to pulmonary fibrosis

作　　者：蔡琳[1] 吴壮[1] 徐军[1] 

机构地区：[1]广州医学院附属第一医院广州呼吸疾病研究所,广东广州510120

出　　处：《医学研究生学报》2008年第6期572-576,I0002,共6页Journal of Medical Postgraduates

基　　金：国家自然科学基金重点项目资助(批准号:30230180)

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。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 TGF-β1 by fluorescence microscopy. After the transfeetion of HMGB34367-peDNA 3.0, the nucleus extracts from the transfeeted cells were subjected to eleetrophoretie mobility shift assay （EMSA） for the detection of thebinding 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.

关 键 词：高迁移率族蛋白B1 Α-平滑肌肌动蛋白 气道重塑 纤维化 

分 类 号：R562[医药卫生—呼吸系统]