Orexin Receptor Spatial Structure Changes As Pointer To Screening Tranquilize Mind Constituents In Traditional Chinese Medicine

Insomnia is a kind of difficulty falling asleep or keeping sleep.With the accelerating pace of modern society,insomnia seriously affects people's work,life and learning.The chemical synthesis of drugs used in the treatment of insomnia,prone to drug resistance,and has strong side effects.So far,a large number of studies show that Chinese medicine extract can promote sleep.A lot of natural medicine composition has good curative effect,little side effect,little dependence and wide medicine sources and other advantages that has become an increasing concern.But these traditional Chinese medicine which components play a role through what the target,how to play the role is not very clear.A series of receptor regulation human's sleep,they mainly include:the Orexin receptor,Melatonin receptor,Serotonin receptor,Histamine receptor,Adenosine receptor,GABAa receptor.In addition to the GABAa receptor,the other five receptors are G protein coupled receptors(GPCR).In this study,two fluorescent groups(eYFP and turquiose)are connected to the orexin receptor(OXR)in different parts of the molecule.The enhance yellow fluorescent protein(eYFP)is connected to the OX1R/OX2R third inner ring,enhance cyan fluorescent protein(turquiose)is connected to the OX1R/OX2R molecule C,to construct pcDNA5/FRT/TO-VSV-GluR5-OX1R-eYFP-turquiose and pcDNA5/FRT/TO-VSV-GluR5-OX2R-eYFP-turquiose plasmids(molecular probes).Determined by gene sequencing,connect into the correctness of the gene sequence.The constructed plasmids were transiently transfected into 293T cells 24h,then serum starvation 24h,with a commercially available OXR agonist orexin A(OXA)to stimulate the cells 5min,collected protein samples for western blot.Western blot detection to the tag of VSV protein and ERK/pERK expression level,the experimental group pERK expression level is higher than control group,preliminary verified the effectiveness of molecular probes.At the same time,the constructed plasmid was transfected into Flp-In T-Rex HEK293 cell which Contains Zeocin resistance genes with pOG44 plasmid.After screening Hygromycin B about two weeks,obtain monoclonal cells which containing OXR molecular probe plasmid gene.Reduce the concentration of Hygromycin B to expand training of monoclonal cells,to establish the induced expression of OXR molecular probe Flp-In T-Rex HEK293 stable cell line.The expression of stable cell line induced by Doxycyclin 24 h,observed the fluorescent yellow and cyan fluorescence by high speed fluorescence microscope.Also after serum starvation treatment and OxA,collect samples of protein,using western blot to confirm the validity of molecular probes.Application of perfusion system and high-speed fluorescence microscopy,using Orexin A to treatment Flp-In T-Rex HEK 293 cells which have been expressed molecule probe.Then to complete the real-time monitoring of changes in the conformation of the receptor by fluorescence resonance energy transfer(FRET),obtain FRET receptor activation curve.On this basis,the use of alcohol extract from the Gastrodia elata,Semen Ziziphi Spinosae to treatment Flp-In T-Rex HEK 293 cells which have been expressed OXR molecule probe,studying the influence of the extract to receptor molecules probe FRET baseline,preliminary screening effective ingredients of traditional Chinese medicine.To complete the study of orexin receptor spatial structure changes as pointer to screening active ingredients in Gastrodia elata,Semen Ziziphi Spinosae etc(mainly with sedation sedative effects of traditional Chinese medicine)Chinese medicine.The main results of this thesis are as follows:(1)To complete the construction of pcDNA5/FRT/TO-VSV-GluR5-OX1R-eYFP-turquiose and pcDNA5/FRT/TO-VSV-GluR5-OX2R-eYFP-turquiose molecular probe plasmid.And after transient transfection by fluorescence detection and western blot test results to determine the constructed OXR molecular probes the effectiveness of the plasmid.(2)After optimized molecular probes,establish induced expression of OXR molecular probe Flp-In T-Rex HEK293 stable cell lines.By western blot test results prove that the effectiveness of the constructed OXR molecular probes plasmid again.(3)Finish the monitoring of the FRET of stable cell lines,and ensuring the effective of stable cell lines by western blot.(4)Preliminary separation and extraction of a sedative effect of traditional Chinese medicine,and extract stimulates cell to examine the influence of the extract of receptor molecules probe FRET baseline,to preliminary screening effective ingredients of traditional Chinese medicine.Conclusion:This study is mainly on the basis of orexin receptor,connect the eYFP and turquiose into orexin receptor,to construct OXR molecular probe plasmid completely.And establish the stability of the induced expression plasmid OXR molecular probe cell line.By fluorescence detection and western blot test determining effectiveness of OXR molecular probes plasmid.Using the characteristics of fluorescence resonance energy transfer technology and the spatial structure change after GPCR binding ligands to establish drug screening platform for screening of traditional Chinese medicine which with high sensitivity completely.Gastrodia elata,Semen Ziziphi Spinosae etc Chinese medicine has carried on the preliminary screening,dedicated to finding targets clear calm the constituents in traditional Chinese medicine.And for the development of Chinese medicine as a lead compound for a class of drugs in the treatment of insomnia provide a technical basis.