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

作　　者：谭晓文[1,2] 董鹏志[1,2] 宁召臣[1,2] 朱彦[1,2] TAN Xiao-wen DONG Peng-zhi NING Zhao-cheng ZHU Yan(Tianjin Key Laboratory of Modern Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China Research and Development Center of Chinese Materia Mediea, Tianjin International Joint Academy of Bioteehnology and Medicine, Tianjin 300457, China)

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》2017年第1期6-10,共5页Journal of Tianjin University of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81274128);国际科技合作项目(2013DFA31620)

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p CMV-h P2Y12-C-His转染入HEK293细胞,采用遗传霉素(G418)筛选获得稳定转染的h P2Y12-C-His-HEK293细胞系,以实时荧光定量聚合酶链式反应(q RT-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。q RT-PCR和Western Blot结果表明P2Y12-His m RNA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P2Y12Lable-free Enspire多功能读板机的无标记高通量筛选。[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] p CMV-h P2Y12-C-His was stably transfected into HEK293 cells with the application of polyethylene imine（PEI） and geneticin（G418）, RTq PCR 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-HisHEK293 stable cell line was successfully obtained with the application of polye-thylene imine（PEI） and geneticin（G418）. RT-q PCR and Western blotting results showed that m RNA 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 were7 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.

关 键 词：ADP受体 无标记 高通量 

分 类 号：R284[医药卫生—中药学]