基于高内涵分析方法建立稳定共表达人δ阿片受体与PKAcat-EGFP的CHO细胞模型

Establishment of CHO cell model of stable co-expression of human delta opioid receptor and PKAcat-EGFP based on high-content analysis screening

目的利用高内涵分析(HCA)方法用中国仓鼠卵巢(CHO)细胞建立人δ阿片受体(h DOR)及增强型绿色荧光蛋白(EGFP)标记的蛋白激酶A催化亚基(PKAcat)融合蛋白(PKAcat-EGFP)稳定共表达的细胞模型,为体外高通量筛选作用于h DOR的药物及药物分子机制研究奠定基础。方法通过脂质体介导法将潮霉素B抗性的h DOR重组质粒(pc DNA3.1/Hygro(+)-h DOR)转染入已稳定表达PKAcat-EGFP的CHO细胞中,随后用含潮霉素B的选择性培养基培养细胞,有限稀释法挑取耐药单克隆,PKA重分布实验筛选阳性克隆,利用Z′因子对建立的细胞模型的可靠性进行评价,利用PKA重分布实验与LANCE c AMP 384试剂盒检测受体功能。结果 PKA重分布实验与LANCE c AMP 384试剂盒检测结果表明,CHO-PKAcatEGFP/h DOR-3号克隆反应性良好,特异性DOR激动剂SNC80 100 nmol·L-1作用时的Z′平均值为0.615,表明该细胞模型可靠,经多次传代后,细胞中的h DOR在40代以内能保持稳定表达,SNC80可浓度依赖性地激活细胞上的h DOR,其EC50值为(6.192±1.225)×10-9mol·L-1。结论成功建立了h DOR与PKAcatEGFP融合蛋白稳定共表达的细胞模型CHO-PKAcat-EGFP/h DOR-3。

OBJECTIVE To establish a cell model which can stably co-express human delta opioid receptor（h DOR） and enhanced green fluorescent protein（EGFP） labeled catalytic domain of c AMPdependent protein kinase A（PKAcat） fusion protein（PKAcat-EGFP） in Chinese hamster ovary（CHO）cells by high-content analysis screening technology in order to contribute to the high-throughput screening of h DOR drugs and research on drug molecular mechanisms in vitro. METHODS Hygromycin B resistant h DOR recombinant plasmid [pc DNA3.1/Hygro（＋）-h DOR] was transfected into CHO cells stably expressing PKAcat-EGFP using a Lipofectin based method. Transfected cells were selected in a culture medium containing hygromycin B. The positive clones were selected by PKA redistribution assay. Z′factor was used for evaluation and validation of the reliability of the cell model. PKA redistribution assay and LANCE cAMP 384 Kit were used to test the function of the receptors in selected clones. RESULTS The CHO-PKAcat-EGFP/h DOR-3 cell model exhibited stable response in PKA redistribution assay and LANCE cAMP 384 Kit. After treatment with SNC80 100 nmol·L^-1 for 30 min, the average value of Z′factor was 0.615, which suggested that the cell model was reliable. The expression of h DOR in the cell model remained stable after a few generations. SNC80 could activate h DOR in a concentration-dependent manner with EC50 of（6.192±1.225）×10^-9 nmol·L^-1. CONCLUSION The CHO-PKAcat-EGFP/h DOR-3 cell model with stable co-expression of h DOR and PKAcat-EGFP is successfully established.