基于Oct4基因启动子活性的高通量药物筛选细胞模型的构建及应用
Construction and application of high-throughput drug screening cell model based on Oct4 gene promoter activity
田银 1李珊珊 2魏明月 3张欢 3汤熙翔4
作者信息
- 1. 福州大学先进制造学院,福建晋江 362200;自然资源部第三海洋研究所,福建厦门 361005
- 2. 自然资源部第三海洋研究所,福建厦门 361005
- 3. 自然资源部第三海洋研究所,福建厦门 361005;广东医科大学,海洋医药研究院,广东湛江 524023
- 4. 福州大学先进制造学院,福建晋江 362200;自然资源部第三海洋研究所,福建厦门 361005;广东医科大学,海洋医药研究院,广东湛江 524023
- 折叠
摘要
目的 以人源Oct4基因启动子为靶点构建双荧光素酶报告基因载体,建立高通量药物筛选细胞模型,筛选能显著上调Oct4基因启动子活性的海洋来源微生物次级代谢产物.方法 将人源Oct4基因启动子序列克隆至萤火虫荧光素酶报告基因载体pGL6-TA,构建重组质粒pGL6-TA-Oct4-luc并与内参质粒pRL-SV40-C瞬时共转染293T细胞,以OAC1作为激活剂,建立双荧光素酶报告基因筛选系统,通过检测相对荧光素酶表达水平的变化反映Oct4基因启动子活性,对309份海洋来源微生物发酵粗提物样品进行活性筛选;CCK-8法检测粗提物对间充质干细胞活力的影响.结果 双酶切和基因测序法证实重组质粒构建正确;将质粒转染293T细胞后,与空载pGL6-TA相比较,重组质粒pGL6-TA-Oct4-luc具有显著启动子活性(P＜0.05);与0 μmol/L相比较,粗提物Z-179-1处理细胞24 h后Oct4基因启动子活性明显升高(P＜0.05),作用质量浓度为10 μg/mL时效果最为显著;CCK-8法检测结果显示Z-179-1显著促进间充质干细胞的增殖,且呈剂量依赖性.结论 成功建立了靶向人源Oct4基因启动子的药物高通量筛选细胞模型,并筛选出海洋来源微生物发酵粗提物Z-179-1,能显著活化Oct4基因启动子,其活性成分值得开展进一步化学研究.
Abstract
Objective A dual luciferase reporter gene vector was constructed by targeting the human Oct4 gene promoter,and a high-throughput drug screening cell model was established to screen marine-derived microbial secondary metabolites that could significantly upregulate the promoter activity of Oct4 gene.Methods The human Oct4 gene promoter sequence was cloned into the firefly luciferase reporter gene vector pGL6-TA,and the recombinant plasmid pGL6-TA-Oct4-luc was constructed and transiently co-transfected with the internal reference plasmid pRL-SV40-C into 293T cells,with OAC1 as the activator,a dual-luciferase reporter gene screening system was constructed,the activity of Oct4 gene promoter was reflected by detecting changes in relative luciferase expression level,and 309 samples of marine-derived microbial fermentation crude extracts were screened for activity;effect of crude extracts on Mesenchymal stem cells(MSCs)viability was measured using CCK-8 assay.Results The recombinant plasmid were successfully constructed by double enzyme digestion and gene sequencing.After the plasmids were transfected into 293T cells,the pGL6-TA-Oct4-luc recombinant plasmid had significant promoter activity compared with pGL6-TA vector(P＜0.05).Compared with 0 μmol/L,the activity of Oct4 gene promoter was significantly increased after 24 h of crude extract Z-179-1 treated cells(P＜0.05),and the effect is most significant at concentration of 10 μg/mL.CCK-8 assay showed that Z-179-1 significantly promoted the proliferation of Mesenchymal stem cells(MSCs)in a dose-dependent manner.Conclusion A high-throughput drug screening cell model targeting human Oct4 gene promoter was successfully constructed,and the marine-derived microbial fermentation crude extract Z-179-1 was screened to significantly activate the activity of Oct4 promoter,and its active components deserved further chemical research.
关键词
干细胞/多能性/Oct4/海洋微生物/药物筛选模型Key words
stem cell/pluripotency/Oct4/marine microorganisms/drug screening modle引用本文复制引用
基金项目
亚洲海洋合作基金(99950410)
福建省科技计划项目(2021N0028)
出版年
2024
NETL
NSTL
万方数据