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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Gα15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)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)-α-Methylserine-O-phosphate(MSOP)>(RS)-α-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系统,适合于mGluR4激动剂/拮抗剂的筛选。
To find a modulator of metabotropic glutamate receptor subtype 4 (mGluR4), a high-throughput screening (HTS) system based on cell functional assays was established by fluorescence detection of intracellular calcium concentration. Human mGluR4 gene was transfected into human embryonic kidney cells (HEK-293) stably expressing Gα15 protein. The cell lines stably expressing mGluR4 were screened by Zeocin, and the biological function of this cell line was confirmed by calcium flow assay. Optimized the incubation time of fluorochrome in the experimental system, the solvent dimethylsulfoxide (DMSO) tolerance, and the solvent sodium hydroxide (NaOH) tolerance, established a reliable and stable screening system. Calcium flow test data indicate that the order of mGluR4 cell line activity on its agonists is: L - (+) - 2-Amino-4-phosphonobutyric acid (L-AP4)> L-Serine- )> L-Glutamicacid (L-Glu); antagonist is: (RS) -α-Methylserine-O-phosphate (MSOP)> (RS) -α-Methyl-4-phosphonophenylglycine (MPPG). In 96 and 384 cell microplates, the Z ’factor of this screening system was found to be 0.80 and 0.65, respectively. The results show that the stable cell line has a stable detection system, suitable for mGluR4 agonist / antagonist screening.