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| ADP receptor P2Y12-targeted screening of TCM anti-thrombotic activities by Lable-free cell phenotype profiling |
| Hits 1594 Download times 1585 Received:October 20, 2016 |
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| DOI
10.11656/j.issn.1673-9043.2017.01.02 |
| Key Words
ADP receptor;lable-free;high throughput |
| Author Name | Affiliation | E-mail | | TAN Xiao-wen | Tianjin Key Laboratory of Modern Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
Research and Development Center of Chinese Materia Medica, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China | | | DONG Peng-zhi | Tianjin Key Laboratory of Modern Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
Research and Development Center of Chinese Materia Medica, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China | pengzhiryn@163.com | | NING Zhao-cheng | Tianjin Key Laboratory of Modern Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
Research and Development Center of Chinese Materia Medica, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China | | | 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 Medica, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China | yan.zhu@tufts.ed |
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| Abstract
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| [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. |
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