以ADP受体P2Y12为靶标的中药抗栓活性无标记细胞表型筛选

ADP receptor P2Y12-targeted screening of TCM anti-thrombotic activities by Lable-free cell phenotype profiling

关键词:  ADP受体  无标记  高通量

Key words:  ADP receptor  lable-free  high throughput

摘要:

[目的] 建立二磷酸腺苷（ADP）受体P2Y12的无标记方法以高通量、低毒性地筛选中药抗血小板/血栓活性物质。[方法] 应用聚乙烯亚胺（PEI）将质粒pCMV-hP2Y12-C-His转染入HEK293细胞，采用遗传霉素（G418）筛选获得稳定转染的hP2Y12-C-His-HEK293细胞系，以实时荧光定量聚合酶链式反应（qRT-PCR）和蛋白免疫印迹（Western Blot）的方法，分别从转录和翻译水平验证P2Y12-His的表达，采用Lable-free Enspire多功能读板机检测激动剂ADP的激动作用，以及抑制剂替卡格雷的抑制作用，以确定筛选体系的可靠性，并利用该体系筛选中药活性组分及单体共30种。[结果] 经PEI转染和G418筛选获得了P2Y12-His-HEK293稳转细胞株。qRT-PCR和Western Blot结果表明P2Y12-His mRNA和蛋白表达量均显著性增加。Lable-free Enspire多功能读板机检测实验表明P2Y12受体激动剂ADP，抑制剂替卡格雷，有显著的激动及抑制作用，且具有明显的量效关系。中药组分及单体筛选结果表明，有7种组分或单体对P2Y12受体具有良好的抑制作用。[结论] 本实验成功建立了ADP受体P2Y12 Lable-free Enspire多功能读板机的无标记高通量筛选。

Abstract:

[Objective] To establish the ADP receptor P2Y12 lable-free, high throughput and low toxicity screening methods in traditional Chinese medicine anti platelet/active substances.[Methods] pCMV-hP2Y12-C-His was stably transfected into HEK293 cells with the application of polyethylene imine (PEI) and geneticin (G418), RT-qPCR and Western blotting were used to proof the expression of P2Y12-His, respectively, from the transcription level and translation level. The agonistic action of ADP and the inhibitory effect of the inhibitor Ticagrelor were detected by Lable-free Enspire multimode detection to ensure the reliability of the screening system. And the system was used to screen the active components of Traditional Chinese Medicine and 30 kinds of monomers.[Results] P2Y12-His-HEK293 stable cell line was successfully obtained with the application of polye-thylene imine (PEI) and geneticin (G418). RT-qPCR and Western blotting results showed that mRNA and protein expression of P2Y12-His were significantly increased. Lable-free Enspire multimode detection results showed that the ADP receptor P2Y12 agonist ADP and inhibitor Ticagrelor had showed an agonistic or inhibitory effect, and had obvious concentration response relationship. The results of screening traditional Chinese medicine components and monomer showed that there were 7 kinds of components or monomers which had good inhibitory effect on P2Y12 receptor.[Conclusion] This ADP receptor P2Y12 lable-free high throughput screening method was established in this study.