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同型半胱氨酸对FABP4启动子活性的影响 预览

Effect of homocysteine on activity of human FABP4 promoter in macrophages
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摘要 目的通过克隆脂肪酸结合蛋白4(FABP4)基因启动子,确定其活性核心区和功能片段,分析心血管疾病独立危险因子同型半胱氨酸(Hcy)对FABP4启动子活性的影响。方法应用生物信息学预测FABP4基因启动子区顺式转录作用元件和反式作用因子,以pGL3-Basic为载体,采用基因重组法构建启动子截取片段,转染HEK-293A细胞,观察不同截取片段的荧光素酶活性变化,确定活性最强的片段。进一步将核心启动子片段(-2000/-1)转染巨噬细胞,观察不同浓度Hcy和DNA甲基化抑制剂5-氮杂胞苷(AZC)对FABP4基因启动子活性的影响。结果不同长度截取片段转染HEK-293A细胞后检测荧光素酶活性结果显示,与pGL3对照组比较,-2000/-1片段转录活性最强。将核心启动子片段(-2000/-1)转染巨噬细胞并用不同浓度Hcy干预后,与0μmol/LHcy组比较,100和200μmol/LHcy组启动活性升高;与100μmol/LHcy组比较,在100μmol/LHcy基础上用AZC干预后,FABP4基因启动活性升高(P<0.05)。结论成功克隆FABP4启动子的4个片段,且确定FABP4基因核心启动子位于-2000/-1片段;Hcy可以促进FABP4基因核心启动子片段活性,补充AZC后可以进一步促进Hcy引起的FABP4启动子活性升高。 Objective To clone the promoter of fatty acid-binding protein 4(FABP4)and determine its core active region and functional fragment,and meanwhile to determine the effect of homocysteine(Hcy),an independent risk factor for cardiac vascular disease,on the activity of FABP4 promoter so as to provide an experimental basis for further research on the function of FABP4.Methods Cis-acting elements and transacting factors were predicted in the promoter region of FABP4 by bioinformatics.The promoter interception fragments were constructed with pGL3-basic vector by gene recombination.The interception fragment with strongest activity was identified by observing the changes in luciferase activity after transfecting different fragments into HEK-293A cells.Meanwhile,after transfection of core promoter fragment(-2000/-1)into macrophages,the effect of different concentrations of Hcy and DNA methylation inhibitor 5-azacytidine(AZC)on the promoter activity of FABP4 gene was detected.Results After transfection of HEK-293A cells with interception fragments of different length,the detection of luciferase activity showed that the transcription activity of-2000/-1 fragment was the strongest in comparison with the pGL3 control group.After transfecting macrophages with core promoter fragment-2000/-1 and treating these cells with different concentrations of Hcy,the promoter activity was significantly increased in the 100 and 200μmol/L Hcy groups compared with the 0μmol/L Hcy group;compared with the 100μmol/L Hcy group,the promoter activity of FABP4 gene was further increased after intervention with AZC on the basis of 100μmol/L Hcy(P<0.05).Conclusions The four fragments of FABP4 promoter have been successfully cloned and the core promoter region of FABP4 is located in the-2000/-1 fragment.Hcy can promote the activity of the core promoter fragment of FABP4 gene,supplementation of AZC can further increase the Hcy-induced activity of FABP4 promoter.
作者 熊建团 李旭生 杨安宁 杨松昊 邓梅 王磊 高源 李南 杨晓玲 贾月霞 姜怡邓 Jian-tuan Xiong;Xu-sheng Li;An-ning Yang;Song-hao Yang;Mei Deng;Lei Wang;Yuan Gao;Nan Li;Xiao-ling Yang;Yue-xia Jia;Yi-deng Jiang(College of Pharmacy,Ningxia Medical University,Yinchuan,Ningxia 750004,China;Department of Orthopedics,General Hospital of Ningxia Medical University,Yinchuan,Ningxia 750004,China;Basic Medical College,Ningxia Medical University,Yinchuan,Ningxia 750004,China)
出处 《中国现代医学杂志》 2018年第29期25-30,共6页 China Journal of Modern Medicine
基金 国家自然科学基金(No:81560084);宁夏自然科学基金(No:NZ16063);宁夏高等学校科学研究项目(No:NGY2016087)。
关键词 FABP4 启动子活性 同型半胱氨酸 FABP4 promoter activity homocysteine macrophage
作者简介 [通信作者]姜怡邓,E-mail:nyxxgjyd@163.com。
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