| 田银,李珊珊,魏明月,等.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[J].中国海洋药物,2024,43(3):37-45. |
| 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 |
| 投稿时间：2022-10-28 修订日期：2023-05-04 |
| DOI：10.13400/j.cnki.cjmd.2024.03.006 |
| 中文关键词: 干细胞 多能性 Oct4 海洋微生物 药物筛选模型 |
| English Keywords:Stem cell Pluripotency Oct4 Marine microorganisms Drug screening modle |
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| 中文摘要: |
| 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。 |
| English Summary: |
| Objective To construct a dual-luciferase reporter gene vector with human Oct4 gene promoter as target, and a high-throughput drug screening cell model was established to screen for secondary metabolites of marine microorganisms that can effectively maintain?the pluripotency
of?stem cells. 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 the relative luciferase expression level, and 309 samples of marine-derived microbial fermentation crude extracts were screened for activity. Results The recombinant plasmid were successfully constructed by double enzyme digestion and gene sequencing；After the plasmids were transfected into 293T cells for 24 h, the pGL6-TA-Oct4-luc recombinant firefly luciferase reporter plasmid had significant promoter activity compared with pGL6-TA group (P<0.05); Compared with 0μmol/L, the activity of Oct4 gene promoter was significantly increased after 24 h of crude extract Z-179-1 treated cells (P<0.05)，the effect is most significant at concentration of 25 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, the fermentation crude extract Z-179-1 of marine-derived fungus Z-179 was screened to significantly activate the activity of Oct4 promoter, and its active components deserve further chemical research. |
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