Construction of Reporter Gene Cell Model for Detection of Histamine Phosphate
OBJECTIVE To establish the cell model of reporter gene for detecting histamine phosphate.METHODS The plas-mids of human histamine phosphate H1 receptor(H1R),H2 receptor(H2R)and the response elements of G protein-coupled receptor alpha subunits,including Gs,Gi or Gq,cloned with reporter gene were co-transfected into HEK293 cells.The transfected ratio of the plasmid was optimized.Subsequently,the expression and function of H1R and H2R were verified by detecting the change of cAMP content and cellular[Ca2+]after the effect of agonists.The chemiluminescence activities of HEK293 cells transfected with H1 R and/or H2R under different concentrations of histamine were compared.Finally,the cell model was verified by adding histamine phosphate into compound amino acid injection,succinyl gelatin injection,and enoxaparin sodium solution to simulate the detection and calculating the recovery rate.RESULTS When the amount of three plasmids was 1∶1∶10,the response value of cells to histamine phosphate was higher,this ratio was used for subsequent transfection.The changes of cAMP and Ca2+contents in cells verified the overexpression of H1R and H2R and the function of reporter gene response element.When the concentration of histamine phosphate was higher than 0.64 nmol·L-1,the chemiluminescence value of cells overexpressing H1R and H2R reporter genes(H1R/H2R-Luc cells),was higher than that of other groups(P＜0.05).This cell model was used to detect the histamine phosphate added in amino acid injection,succinyl gelatin injection,and enoxaparin sodium solution.The recovery rates were between 88％-121％when the concentration of histamine phosphate was between 3.2-400 nmol·L-1.CONCLUSION The H1R/H2R-Luc cells constructed successfully in the present study would be used for the detection of histamine phosphate.
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