| 基于荧光共振能量转移技术的STAT3二聚化抑制剂筛选模型的建立 |
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| 引用本文： | 陈俊生,赵麟,李静,戚欣. 基于荧光共振能量转移技术的STAT3二聚化抑制剂筛选模型的建立[J]. 中国海洋药物, 2018, 37(3): 9-17 |
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| 作者姓名： | 陈俊生 赵麟 李静 戚欣 |
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| 作者单位： | 中国海洋大学医药学院，海洋国家实验室海洋药物与生物制品功能实验室,中国海洋大学医药学院，海洋国家实验室海洋药物与生物制品功能实验室,中国海洋大学医药学院，海洋国家实验室海洋药物与生物制品功能实验室,中国海洋大学医药学院，海洋国家实验室海洋药物与生物制品功能实验室 |
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| 基金项目： | 青岛海洋科学与技术国家实验室鳌山科技创新计划项目(No.2015ASKJ02) |
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| 摘 要： | 目的%20构建STAT3二聚化抑制剂筛选模型,为STAT3抑制剂筛选提供实验方法。方法%20分别构建pECFP-N1-STAT3和pEYFP-N1-STAT3的荧光报告载体,利用脂质体转染技术将二者共转入HEK-293T细胞,利用ECFP和EYFP两种荧光蛋白之间能量共振转移,检测磷酸化STAT3分子的二聚化水平,并检测Stattic对二聚化的影响。结果%20成功构建了pECFP-N1-STAT3和pEYFP-N1-STAT3的荧光报告载体。将两种荧光报告载体共转染HEK-293T细胞后,Western%20Blot%20检测结果显示STAT3以及p-STAT3的表达水平明显增加。以458nm波长激发ECFP,其发射波长可激发EYFP。加入STAT3二聚化抑制剂Stattic后,荧光强度降低,且呈现一定的剂量依赖性。结论%20基于荧光共振能量转移技术的STAT3二聚化抑制剂筛选模型构建成功。
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| 关 键 词： | 信号传导与转录激活因子3(STAT3) 二聚化 荧光共振能量转移(FRET) Stattic |
| 收稿时间： | 2017-12-19 |
| 修稿时间： | 2018-03-05 |
Establishment%20of%20the%20screening%20model%20for%20STAT3%20dimerization%20inhibitor%20based%20on%20fluorescence%20resonance%20energy%20transfer%20technology |
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| CHEN Jun-sheng,ZHAO Lin,LI Jing and QI Xin. Establishment%20of%20the%20screening%20model%20for%20STAT3%20dimerization%20inhibitor%20based%20on%20fluorescence%20resonance%20energy%20transfer%20technology[J]. Chinese Journal of Marine Drugs, 2018, 37(3): 9-17 |
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| Authors: | CHEN Jun-sheng ZHAO Lin LI Jing QI Xin |
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| Affiliation: | School of Medicine and Pharmacy,Ocean University of China,Laboratory of Marine Drugs and Biological Products,Qingdao National Laboratory of Marine Science and Technology,School of Medicine and Pharmacy,Ocean University of China,Laboratory of Marine Drugs and Biological Products,Qingdao National Laboratory of Marine Science and Technology,School of Medicine and Pharmacy,Ocean University of China,Laboratory of Marine Drugs and Biological Products,Qingdao National Laboratory of Marine Science and Technology,School of Medicine and Pharmacy,Ocean University of China,Laboratory of Marine Drugs and Biological Products,Qingdao National Laboratory of Marine Science and Technology |
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| Abstract: | Abstract:%20Objective to construct%20a%20screening%20model%20of%20STAT3%20dimerization%20inhibitor%20and to provide%20a%20novel%20experimental%20method%20for%20STAT3%20inhibitor%20screening.%20Methods%20Two%20fluorescence%20reporter%20vectors,%20pECFP-N1-STAT3%20and%20pEYFP-N1-STAT3,%20were%20constructed%20by%20molecular%20biological%20technique;%20Liposome-mediated%20transfection%20technique%20was%20used to transfer%20gene%20into%20HEK-293T%20cells;%20The%20mutual%20function%20between%20ECFP%20and%20EYFP%20was%20confirmed%20using%20the%20energy%20resonance%20transfer%20technology%20by%20which%20the%20dimerization%20level%20of%20STAT3%20proteins%20was%20measured;The%20effect%20of%20Stattic%20on%20dimerization%20of%20STAT3%20proteins%20was%20examined%20in%20this%20model.%20Results%20The%20fluorescent%20reporter%20vectors%20containing%20the%20STAT3%20gene%20were%20successfully%20constructed.%20Western%20Blot%20showed%20that%20the%20expression%20levels%20of%20STAT3%20and%20phosphorylated%20STAT3%20proteins%20were%20significantly%20increased%20after%20transfection.%20The%20ECFP%20was%20excited%20at%20a%20wavelength%20of%20458%20nm%20and%20its%20emission%20wavelength%20could%20excite%20EYFP.%20The%20addition%20of%20STAT3%20dimerization%20inhibitor%20Stattic%20induced%20the%20reduction%20of%20fluorescence%20intensity%20in%20dose-dependent%20manner,%20suggesting%20that%20Stattic%20inhibited%20STAT3%20dimerization%20in%20this%20model.%20Conclusion%20The%20screening%20model%20of%20STAT3%20dimerization%20inhibitor%20is%20established%20successfully%20based%20on%20FRET%20technology. |
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