一种改良的5-羟色胺转运体活性化合物高内涵筛选方法

An improved high content screening method for compounds on activity of serotonin transporters

关键词:  5-羟色胺转运体  APP⁺  hSERT-HEK293细胞系  高内涵筛选

Key words:  SERT  APP⁺  hSERT-HEK293 cell line  HCS

摘要:

[目的] 建立一种改良的5-羟色胺转运体（SERT）活性化合物高内涵筛选（HCS）方法，包括构建稳定转染hSERT细胞株和优化SERT活性化合物HCS条件。[方法] 应用聚乙烯亚胺（PEI）将质粒hSERT-pcDNA3转染入HEK293细胞，采用遗传霉素（G418）筛选获得稳定转染细胞系，以实时荧光定量聚合酶链式反应（Real-time PCR）和蛋白免疫印迹（Western blotting）方法分别从转录和翻译水平验证hSERT表达，采用SERT荧光底物4-（4-二甲氨基）苯基-1-甲基吡啶（APP⁺）在高内涵系统上验证SERT摄取功能并优化其检测条件，应用选择性SERT抑制剂Fluoxetine和SERT增强剂Tianeptine验证SERT HCS方法可靠性。[结果] 经PEI转染和G418筛选获得了稳定转染hSERT-HEK293细胞株。与HEK293细胞相比，hSERT-HEK293细胞中hSERT mRNA和蛋白表达量均显著性增加，APP⁺摄取实验表明hSERT-HEK293细胞摄取APP⁺显著性增加，并可被Fluoxetine特异性抑制。实验优化了高内涵检测APP⁺摄取实验中APP⁺浓度、孵育时间、数据分析方法等条件。Fluoxetine显著地抑制hSERT-HEK293对APP⁺的摄取，Tianeptine显著地增强hSERT-HEK293对APP⁺的摄取，证实此HCS方法的可靠性。[结论] 本研究首次优化了应用APP⁺检测SERT活性的HCS条件，显著提高了SERT荧光检测方法灵敏度，并首次证实此HCS方法适用于SERT促进剂筛选，因此，建立了一种改良的SERT活性化合物HCS方法。

Abstract:

[Objective] To establish an improved high content screening (HCS) method for compounds on activity of serotonin transporter (SERT), a stably transfected human SERT cell line will be obtained and the test conditions of HCS method for SERT activity will be optimized. [Methods] To obtain the stably transfected SERT cell line, plasmid hSERT-pcDNA3 was transfected into the HEK293 cell line by polyethylenimine (PEI) and then the cells were screened by antibiotic G418. To verify the quality of this stably transfected cell line, expression of hSERT mRNA and protein was tested by Real-time PCR and Western blotting, respectively. To test the SERT reuptake activity, 4-(4-(Dimethylamino)phenyl)-1-methylpyridinium (APP⁺), a fluorescent substrate of SERT, was used and the test conditions of HCS assay were optimized. The positive drugs including selective serotonin reuptake inhibitor fluoxetine and enhancer tianeptine were used to confirm the reliability of this HCS assay. [Results] The stably tansfected hSERT-HEK293 cell line was obtained by PEI transfection and G418 screening. Compared to the HEK293 cells, expression of hSERT mRNA and protein in hSERT-HEK293 cells was significantly increased, and its transport ability of APP⁺ was also significantly increased, which could be specifically blocked by fluoxetine. The test conditions of HCS for SERT activity were optimized including the concentration of APP⁺, its incubation time and the method for data analysis. The SERT activity of hSERT-HEK293 cells were significantly inhibited by fluoxetine while significantly enhanced by tianeptine, which confirmed the reliability of this HCS method. [Conclusion] This study firstly optimized the HCS assay with APP⁺ in testing SERT activity, which obviously improved the sensitivity of HCS method for SERT activity, and it is the first time to show this HCS method suitable for screening SERT enhancers, so this study established an improved HCS method for compounds on hSERT activity.