摘要
目的探索用于磷酸组胺检测的报告基因细胞模型构建。方法将人磷酸组胺H1受体(H1R)、H2受体(H2R)的过表达质粒,以及报告基因的G蛋白偶联受体亚型Gs、Gi和Gq响应元件的载体单独或共同转染至HEK293细胞,并对质粒的转染比例进行优化;检测H1R或H2R激动剂作用后细胞内cAMP和Ca^(2+)含量的变化验证受体的表达和功能;对不同细胞模型经组胺作用后的化学发光值进行比较;通过在复方氨基酸注射液、琥珀酰明胶注射液以及依诺肝素钠中添加磷酸组胺进行模拟检测,计算回收率以验证准确性。结果共转染时3种质粒的比例为1∶1∶10时,细胞对磷酸组胺的响应值较高,后续采用此比例进行转染;对细胞内cAMP和Ca^(2+)含量的变化测定验证了H1R和H2R的过表达和功能;其中过表达H1R和H2R的报告基因细胞(H1R/H2R-Luc细胞)与单独过表达H1R或H2R的细胞相比,当磷酸组胺浓度高于0.64 nmol·L^(-1)时,其化学发光值均高于其他组细胞(P<0.05);用该模型检测复方氨基酸注射液、琥珀酰明胶注射液以及依诺肝素钠中添加的磷酸组胺,当磷酸组胺含量在3.2~400 nmol·L^(-1)内时呈现良好的线性关系,磷酸组胺的回收率在88%~121%。结论构建的H1R/H2R-Luc细胞可用于磷酸组胺的检测。
OBJECTIVE To establish the cell model of reporter gene for detecting histamine phosphate.METHODS The plasmids 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 after the effect of agonists.The chemiluminescence activities of HEK293 cells transfected with H1R 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 Ca^(2+)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.
作者
陈莉
陶禹
霍家燕
季文君
顾晓红
潘尔卓
陶金
陈卫
CHEN Li;TAO Yu;HUO Jiayan;JI Wenjun;GU Xiaohong;PAN Erzhuo;TAO Jin;CHEN wei(Suzhou Institute for Drug Control,Suzhou 215104,China;Soochow University,Suzhou 215031,China;Shanghai VKEY Biotechnologies Co.Ltd,Shanghai 200241,China)
出处
《中国药学杂志》
CAS
CSCD
北大核心
2024年第11期984-989,共6页
Chinese Pharmaceutical Journal
基金
江苏省市场监督管理局科技计划项目资助(KJ2022038)。
