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1、代谢型谷氨酸受体4亚型(mGluR4)高通量药物筛选模型的建立张娅玲1,2,白艳秋1,21中国科学院北京基因组研究所,北京1013002中国科学院研究生院,北京100039摘要:为发现代谢型谷氨酸受体4亚型(mGluR4)的调节剂,通过荧光检测胞内钙浓度的方法,建立一个基于细胞功能性检测的高通量筛选(HTS)系统。将人mGluR4基因转染稳定表达Ga15蛋白的人胚肾细胞(HEK-293),用Zeocin筛选获得稳定表达mGluR4的细胞株,并通过钙流检测试验证实该细胞系的生物学功能。优化了实验系统中荧光染料的孵育时间,溶剂二甲基亚砜(DMSO)耐受性,以及溶剂氢氧化钠(NaOH)
2、耐受性,建立了可靠稳定的筛选系统。钙流检测试验数据表明,mGluR4细胞系对其激动剂的活性程度排序是:L-(+)-2-Amino-4-phosphonobutyricacid(L-AP4)>L-Serine-O-phosphate(L-SOP)>L-Glutamicacid(L-Glu);拮抗剂是:(RS)-a-Methylserine-O-phosphate(MSOP)>(RS)-a-Methyl-4-phosphonophenylglycine(MPPG)。在96和384细胞微孔培养板中,得到该筛选系统Z’因子分别是0.80和0.65。结果表明,该稳定细胞系拥有一个稳定的检测
3、系统,适合于mGluR4激动剂/拮抗剂的筛选。关键词:代谢型谷氨酸受体4亚型(mGluR4),高通量筛选(HTS),Ga15,激动剂/拮抗剂Developmentofafunctionalcell-basedHTSassayfortheidentificationmGluR4modulatorsYalingZhang1,2,andYanqiuBai1,21BeijingInstituteofGenomics,ChineseAcademyofSciences,Beijing101300,China2GraduateUniversityoftheChineseAcademyofSci
4、ences,Beijing100039,ChinaAbstract:Toidentifymetabotropicglutamatereceptor4(mGluR4)modulatorsbyCa2+influxassay,wedevelopedthefunctionalcell-basedhighthroughput-screening(HTS)assay.ThehumanmGluR4cDNAwastransfectedintoHEK-293stablyexpressingpromiscuousG-protein(Ga15)cells.RecombinantstablemGluR4
5、celllinewasselectedunderZeocinandvalidatedbyCa2+influxassay.TheassaywasoptimizedonloadingtimeofFluoCalciumIndicator,Dimethylsulfoxide(DMSO)toleranceandsodiumhydroxide(NaOH)toleranceusingagonist(L-Glutamicacid(L-Glu))ofmGluR4.TherankorderoftheagonistpotencyforthestablehumanmGluR4celllinewasL-(
6、+)-2-Amino-4-phosphonobutyricacid(L-AP4)>L-Serine-O-phosphate(L-SOP)>L-Glu,andoftheantagonistpotencywas(RS)-a-Methylserine-O-phosphate(MSOP)>(RS)-a-Methyl-4-phosphonophenylglycine(MPPG).Z’factorvalueofthecelllinein96-and384-wellplateformatwas0.80and0.65.Ourdataindicateasuccessfuldevelopmentof
7、functionalhumanmGluR4recombinantstablecelllinethatwassuitableforhighthroughputscreeningtoidentifymGluR4agonist/antagonist.Keywords:metabotropicglutamatereceptor4(mGluR4),highthroughputscreening(HTS),Ga15,agonist/antagonist463/7谷氨酸是公认的最重要的兴奋性神
