TNF-α3′-UTR双荧光素酶报告基因系统的构建及丹参酮类药物筛选  

Construction of p GL3-TNF-α 3'UTR luciferase reporter gene and tanshinone compounds screening

作　　者：韦忠红[1,2] 朱智杰[1,2] 刘玉萍[1,2] 刘兆国[1,2] 盛晓波[1,2] 汪思亮[1,2] 陶丽[1,2] 祝娉婷[1,2] 陈文星[1,2] 王爱云[1,2] 陆茵[1,2] 

机构地区：[1]南京中医药大学药学,江苏南京210046 [2]江苏省药效与安全性评价重点实验室,江苏南京210046

出　　处：《中国药理学通报》2015年第1期77-81,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81173174,81202655);教育部博士点基金(No 20113237110008);江苏高校优势学科建设工程资助项目

摘　　要：目的构建并鉴定p GL3-TNF-α3'端非翻译区(UTR)双荧光素酶报告基因(DLR)表达系统,以此基于调控TNF-α转录后水平对丹参酮类成分进行筛选。方法反转录人脐静脉内皮细胞HUVEC的mRNA成c DNA,以之为模板,PCR扩增含TNF-α3'-UTR的DNA片段全长,经酶切后连接至荧光素酶报告载体p GL3-control上,构建出p GL3-TNF-α3'UTR全长的荧光素酶报告基因载体并进行鉴定。将所构建的p GL3-TNF-α3'UTR与p SVRenilla质粒组成双荧光素酶报告系统共转染至单核巨噬细胞RAW264.7,经脂多糖诱导后利用该双荧光素酶报告基因表达系统判定丹参酮类成分对TNF-α转录后调控是否有影响。结果成功构建p GL3-TNF-α3'UTR荧光素酶报告基因,克隆获得的DNA片段大小及序列与Genbank报道的一致。脂多糖(LPS)可以明显诱导转入p GL3-TNF-α3'UTR载体细胞的荧光强度。丹参酮类成分中隐丹参酮可以明显降低LPS诱导的p GL3-TNF-α3'UTR载体细胞的荧光素酶活性。结论成功构建含TNF-α3'UTR区的双荧光素酶报告基因表达系统,并以此从丹参酮类化合物中筛选出隐丹参酮,可以对TNF-α的转录后水平进行抑制调控。Aim To screen the potential inhibitors of post-transcriptional activity of pro-inflammatory media-tor TNF-α from the lipophilic constituents in Chinese Medicine Salvia miltiorrhiza Bunge （ Danshen） , we es-tablished dual luciferase reporter gene system pGL3-TNF-α3′UTR （ 3′untranslated region ） co-transfected with Renilla control gene. Methods Complementary DNA （ cDNA） template was obtained from human um-bilical vein endothelial cells （ HUVECs ） . The full length DNA of TNF-α 3′-UTR was amplified through PCR, and then connected the luciferase reporter vector pGL3-control after enzyme digestion. pGL3-TNF-α 3′UTR constructs were co-transfected with pSVRenilla into the mononuclear macrophages RAW264. 7 cells. The relative activity of reporter genes was measured by dual luciferase reporter （ DLR ） assay system after the stimulus of lipopolysaccharide （ LPS ） in presence or absence of tanshinones compounds. Results The＆amp;nbsp;pGL3-TNF-α3′UTR luciferase reporter gene was suc-cessfully constructed. The cloning DNA fragment and sequence were both consistent with the GENBANK da-tabase. LPS significantly induced the relative reporter activityof RAW264 . 7 cells transfected with pGL3-TNF-α 3′UTR. Among four tanshinones compounds, we found only cryptotanshinone could significantly de-crease LPS-induced relative reporter activity. Conclu-sion The pGL3-TNF-α 3′UTR construct combined with DLR assay system was successfully established, which can be used to discover the agents such as cryp-totanshinone that regulate the post-transcription of TNF-α in treatment of inflammatory and malignant dis-eases.

关 键 词：TNF-Α 3′非编码区 双荧光素酶报告基因 转录后调控 隐丹参酮 药物筛选 

分 类 号：R284.1[医药卫生—中药学] R329.2[医药卫生—中医学]