东南大学学报（医学版）
東南大學學報（醫學版）
동남대학학보（의학판）
JOURNAL OF SOUTHEAST UNIVERSITY(MEDICAL SCIENCE EDITION)
2014年
2期
133-137
,共5页
高原%邹阳%于影%林金德%张春丽%温传俊%沈干
高原%鄒暘%于影%林金德%張春麗%溫傳俊%瀋榦
고원%추양%우영%림금덕%장춘려%온전준%침간
miR-140生物传感器%荧光素酶%实时荧光定量聚合酶链反应%成软骨诱导
miR-140生物傳感器%熒光素酶%實時熒光定量聚閤酶鏈反應%成軟骨誘導
miR-140생물전감기%형광소매%실시형광정량취합매련반응%성연골유도
miR-140 sensor%luciferase%RT-qPCR%chondrogenesis
目的：利用双荧光素酶报告基因系统构建可检测miR-140活性的生物传感器。方法：首先，在psiCHECK-2双荧光报告基因质粒的多克隆位点插入miR-140成熟体的4个拷贝反义序列，构建miR-140 sensor。其次，将miR-140 sensor和miR-140 mimics共转染HEK-293 T细胞，利用双荧光素酶报告基因系统检验miR-140 sensor的功能。最后，转染miR-140 sensor至大鼠骨髓间充质干细胞（ rat MSCs ），分析在成软骨诱导中miR-140的活性变化，并与miR-140的表达水平相比较。结果：在HEK-293T细胞中，相对于阴性对照组，miR-140 sensor与miR-140 mimics共转能明显降低49％（20 nmol· L-1）和65％（50 nmol· L-1）的荧光活性。将转染miR-140 sensor的rat MSCs成软骨诱导7 d后，荧光活性降低43％，提示miR-140活性升高，与RT-qPCR法检测的miR-140表达水平相一致。结论：构建的miR-140 sensor是一种简单方便有效的miRNA传感器，可用于检测miR-140活性。
目的：利用雙熒光素酶報告基因繫統構建可檢測miR-140活性的生物傳感器。方法：首先，在psiCHECK-2雙熒光報告基因質粒的多剋隆位點插入miR-140成熟體的4箇拷貝反義序列，構建miR-140 sensor。其次，將miR-140 sensor和miR-140 mimics共轉染HEK-293 T細胞，利用雙熒光素酶報告基因繫統檢驗miR-140 sensor的功能。最後，轉染miR-140 sensor至大鼠骨髓間充質榦細胞（ rat MSCs ），分析在成軟骨誘導中miR-140的活性變化，併與miR-140的錶達水平相比較。結果：在HEK-293T細胞中，相對于陰性對照組，miR-140 sensor與miR-140 mimics共轉能明顯降低49％（20 nmol· L-1）和65％（50 nmol· L-1）的熒光活性。將轉染miR-140 sensor的rat MSCs成軟骨誘導7 d後，熒光活性降低43％，提示miR-140活性升高，與RT-qPCR法檢測的miR-140錶達水平相一緻。結論：構建的miR-140 sensor是一種簡單方便有效的miRNA傳感器，可用于檢測miR-140活性。
목적：이용쌍형광소매보고기인계통구건가검측miR-140활성적생물전감기。방법：수선，재psiCHECK-2쌍형광보고기인질립적다극륭위점삽입miR-140성숙체적4개고패반의서렬，구건miR-140 sensor。기차，장miR-140 sensor화miR-140 mimics공전염HEK-293 T세포，이용쌍형광소매보고기인계통검험miR-140 sensor적공능。최후，전염miR-140 sensor지대서골수간충질간세포（ rat MSCs ），분석재성연골유도중miR-140적활성변화，병여miR-140적표체수평상비교。결과：재HEK-293T세포중，상대우음성대조조，miR-140 sensor여miR-140 mimics공전능명현강저49％（20 nmol· L-1）화65％（50 nmol· L-1）적형광활성。장전염miR-140 sensor적rat MSCs성연골유도7 d후，형광활성강저43％，제시miR-140활성승고，여RT-qPCR법검측적miR-140표체수평상일치。결론：구건적miR-140 sensor시일충간단방편유효적miRNA전감기，가용우검측miR-140활성。
Objective:To construct a dual luciferase reporter system based sensor for detecting miR-140 activity. Methods:Firstly, we inserted four copies of miR-140-5p complementary sequences into the multiple cloning site (MCS) of psiCHECK-2 luciferase reporter vector to construct miR-140 sensor.Subsequently, miR-140 sensor and miR-140 mimics were co-transfected into HEK-293T cells and its function was validated by luciferase reporter assay . Finally, miR-140 sensor was also transfected into rat bone marrow stromal cells ( rat MSCs ) and then miR-140 activity was analyzed comparatively with the expression of miR-140 in chondrogenesis .Results:49% (20 nmol · L-1 ) and 65%(50 nmol· L-1 ) reporter activity was decreased in HEK-293T cells relative to NC, when miR-140 sensor was co-transfected with miR-140 mimics.An apparent decrease in reporter activity (43%) was observed at 7 days after chondrogenic induction of rat MSCs compared with control , which reflected an increased miR-140 activity and was consistent with the expression of miR-140 by RT-qPCR method .Conclusion:miR-140 sensor is proved to be an easy , convenient and effective miRNA sensor for detecting miR-140 activity.
