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

作　　者：刘艳庭[1,2] 应树松[1,2] 武艺静 董鹏志[1,2] 吴红华[1,2] 徐砚通[1,2] 

机构地区：[1]天津中医药大学天津市现代中药重点实验室,天津300193 [2]天津国际生物医药联合研究院中药新药研发中心,天津300457

出　　处：《天津中医药大学学报》2016年第3期180-186,共7页Journal of Tianjin University of Traditional Chinese Medicine

基　　金：国家"重大新药创制"科技重大专项(2012ZX09304007);国家自然科学基金青年基金(81403132);天津市高等学校科技发展基金计划项目(20140203);天津市应用基础及前沿技术研究计划项目(12JCYBJC32400)

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h SERT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h SERT-pc DNA3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h SERT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h SERT-HEK293细胞株。与HEK293细胞相比,h SERT-HEK293细胞中h SERT m RNA和蛋白表达量均显著性增加,APP+摄取实验表明h SERT-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h SERT-HEK293对APP+的摄取,Tianeptine显著地增强h SERT-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。[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 h SERT-pc DNA3 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 h SERT m RNA 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 h SERT-HEK293 cell line was obtained by PEI transfection and G418 screening. Compared to the HEK293 cells, expression of h SERT mRNA and protein in h SERT-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 h SERT-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 h SERT activity.

关 键 词：5-羟色胺转运体 APP+ H SERT-HEK293细胞系 高内涵筛选 

分 类 号：R282.7[医药卫生—中药学]