The Sensitization Effect Of Cholic Acid And Geniposide In Qingkailing Injection Was Studied Based On Nucleic Acid Aptamer Technology

ObjectiveIn this study,Qingkailing injection was used as the research object.The combination of nucleic acid aptamers and immunomagnetic beads separation technique was used to obtain Qingkailing injection after cholic acid and geniposide respectively.Then,through the cell model.The nature of the allergic reaction,through the animal model to study the nature of allergic reactions,aimed at clarifying the cholic acid and geniposide in Qingkailing injection caused by allergic reactions and allergic reactions in the role.MethodsThis study mainly includes three parts:the first,labeled with biotin nucleic acid aptamer and streptavidin-coated beads coupled,the conjugate and Qing Kai Ling injection reaction incubation,and then Using magnetic separation technology to remove the target molecules,to obtain the removal of Qingkailing injection.Optimize the experimental conditions,remove as many target molecules as possible.Second,the use of RBL-2H3 cells for hypersensitivity experiments,compared with the target molecules before and after removal of Qingkailing injection of cells on the class of allergic effects,to determine whether the target molecules lead to Qing Kai Ling injection of allergic reactions the reason.Third,the use of rat systemic active allergy test and skin passive allergic reaction model,compared to remove the target molecules before and after the sensitization of Qingkailing injection,and further verify whether the target molecules for Qingkailing injection sensitization of the main reasons.Result1.The Kd values of the three cholic acid aptamers:17.76?M for aptl,8.77M for apt2 and8.93?M for apt3;The Kd of the aptamer N2 was 6.06 ?M.2.In the reaction of the nucleic acid aptamer and the magnetic bead,1 ml of the magneticbeads reacted with different concentrations of the cholic acid aptamer,and thecorresponding amount of 2,M of the aptamer was the highest in the amount of 0.851 nmol;2?M of the aptamer with different volume of magnetic beads reaction,magnetic beads 2ml when coupled with the highest fit,can almost completely fit the appropriate ligand.3.In the three cholic acid aptamers,the apt3 aptamer was coupled with the magnetic beads with a relatively high coupling rate of 42.94%.Among the three different brands of beads,the coupling of the Magbeads beads The maximum amount of 100?l can be coupled to 0.8724nmol of the aptamer.4.The concentration of cholic acidin Qingkailing injection,measured by high-performance liquid phase detection,is about 3214.884?g/ml,in which geniposide is about 446.775?g/ml,in line with the standard in the Pharmacopoeia.In the exclusion of cholic acid experiment,Qingkailing injection dilution in the original liquid,the largest amount of rejection,can remove 376.448nmol of cholic acid.When the volume of the emblem was 1 ml,the rejection rate of cholic acid was 11.12%when the volume of Qingkailing injection was 300?l.5.In cell allergic reactions,Qingkailing injection can cause cell degranulation effect,and remove the cholic acid and geniposide Qingkailing injection on the role of cells was significantly reduced.6.In animal allergy experiments,compared with the normal group,the Qingkailing injection can cause active hypersensitivity in SD rats,and remove the cholic acid and geniposide after Qingkailing injection caused by active allergic rats The effect of the reaction was significantly weakened;in the passive skin allergy test,compared with the normal group,Qingkailing injection can cause passive allergic reactions in rats,and exclusion of cholic acid and geniposide Qingkailing injection caused by the role of passive allergic reactions were significantly decreased.Conclusions1.In this experiment,the Kd values of the nucleic acid aptamers used to remove target molecules from Qingkailing injection were all below 10?M and had high affinity.However,due to the existing technology and reaction conditions and other reasons,can not achieve 100%rejection of the target molecule purposes.2.Qingkailing injection can induce RBL-2H3 cells to produce degranulation effect,the cell morphology changes,and the release of?-aminoglucosidase and tumor necrosis factor-a and other media.Removal of part of the cholic acid and geniposide Qingkailing injection caused by cell degranulation effect was significantly reduced,indicating that cholic acid and geniposide may be caused by Qingkailing injection caused by the main components of allergic reactions.3.Qingkailing injection can lead to active hypersensitivity in SD rats and skin passive reaction,remove part of the cholic acid and geniposide after Qingkailing injection caused by active allergic reactions and skin passive reaction significantly decreased,indicating that Cholic acid and geniposide may be the main component of allergic reactions caused by Qingkailing injection.