中国药理学通报
中國藥理學通報
중국약이학통보
CHINESE PHARMACOLOGICAL BULLETIN
2015年
1期
77-81
,共5页
韦忠红%朱智杰%刘玉萍%刘兆国%盛晓波%汪思亮%陶丽%祝娉婷%陈文星%王爱云%陆茵
韋忠紅%硃智傑%劉玉萍%劉兆國%盛曉波%汪思亮%陶麗%祝娉婷%陳文星%王愛雲%陸茵
위충홍%주지걸%류옥평%류조국%성효파%왕사량%도려%축빙정%진문성%왕애운%륙인
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
TNF-α%3′非編碼區%雙熒光素酶報告基因%轉錄後調控%隱丹參酮%藥物篩選
TNF-α%3′비편마구%쌍형광소매보고기인%전록후조공%은단삼동%약물사선
TNF-α,3′UTR%dual luciferase reporter%post-transcriptional regulation%cryptotanshinone%com-pounds screening
目的：构建并鉴定pGL3-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cDNA,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pGL3-control上,构建出pGL3-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pGL3-TNF-α3′UTR与pSVRenilla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 pGL3-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pGL3-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pGL3-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。
目的：構建併鑒定pGL3-TNF-α3′耑非翻譯區( UTR)雙熒光素酶報告基因( DLR)錶達繫統,以此基于調控TNF-α轉錄後水平對丹參酮類成分進行篩選。方法反轉錄人臍靜脈內皮細胞HUVEC的mRNA成cDNA,以之為模闆,PCR擴增含TNF-α3′-UTR的DNA片段全長,經酶切後連接至熒光素酶報告載體pGL3-control上,構建齣pGL3-TNF-α3′UTR全長的熒光素酶報告基因載體併進行鑒定。將所構建的pGL3-TNF-α3′UTR與pSVRenilla質粒組成雙熒光素酶報告繫統共轉染至單覈巨噬細胞RAW264.7,經脂多糖誘導後利用該雙熒光素酶報告基因錶達繫統判定丹參酮類成分對TNF-α轉錄後調控是否有影響。結果成功構建 pGL3-TNF-α3′UTR熒光素酶報告基因,剋隆穫得的DNA片段大小及序列與Genbank報道的一緻。脂多糖( LPS )可以明顯誘導轉入pGL3-TNF-α3′UTR載體細胞的熒光彊度。丹參酮類成分中隱丹參酮可以明顯降低LPS誘導的pGL3-TNF-α3′UTR載體細胞的熒光素酶活性。結論成功構建含TNF-α3′UTR區的雙熒光素酶報告基因錶達繫統,併以此從丹參酮類化閤物中篩選齣隱丹參酮,可以對TNF-α的轉錄後水平進行抑製調控。
목적：구건병감정pGL3-TNF-α3′단비번역구( UTR)쌍형광소매보고기인( DLR)표체계통,이차기우조공TNF-α전록후수평대단삼동류성분진행사선。방법반전록인제정맥내피세포HUVEC적mRNA성cDNA,이지위모판,PCR확증함TNF-α3′-UTR적DNA편단전장,경매절후련접지형광소매보고재체pGL3-control상,구건출pGL3-TNF-α3′UTR전장적형광소매보고기인재체병진행감정。장소구건적pGL3-TNF-α3′UTR여pSVRenilla질립조성쌍형광소매보고계통공전염지단핵거서세포RAW264.7,경지다당유도후이용해쌍형광소매보고기인표체계통판정단삼동류성분대TNF-α전록후조공시부유영향。결과성공구건 pGL3-TNF-α3′UTR형광소매보고기인,극륭획득적DNA편단대소급서렬여Genbank보도적일치。지다당( LPS )가이명현유도전입pGL3-TNF-α3′UTR재체세포적형광강도。단삼동류성분중은단삼동가이명현강저LPS유도적pGL3-TNF-α3′UTR재체세포적형광소매활성。결론성공구건함TNF-α3′UTR구적쌍형광소매보고기인표체계통,병이차종단삼동류화합물중사선출은단삼동,가이대TNF-α적전록후수평진행억제조공。
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 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.
