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作 者:隋宁 张子伟 胡丹丹 SUI Ning;ZHANG Ziwei;HU Dandan(Beijing Ap Biotechnology Co.,Ltd.,Beijing 100176,China)
机构地区:[1]北京爱思益普生物科技股份有限公司细胞部,北京100176
出 处:《临床研究》2023年第6期4-9,共6页Clinical Research
基 金:医药创新品种及平台培育(Z211100002521003)。
摘 要:目的 建立一种基于细胞水平的第二信使cAMP检测方法来评估化合物对多巴胺(DA)受体的活性影响。方法 通过构建DA受体真核表达载体,应用Flp-In系统和脂质体转染法构建表达DA受体的中国仓鼠卵巢(CHO)细胞系,反转录PCR(RT-PCR)鉴定DA受体的表达情况;利用TR-FRET cAMP方法检测阳性化合物对DA靶点的激活和抑制作用;毛喉素(Forskolin)是一种有效的腺苷酸环化酶(AC)激活剂,诱导细胞内cAMP的形成。结果 经RTPCR鉴定,宿主细胞中无目的表达,而转染后的细胞多巴胺细胞系中有目的基因表达,成功构建表达多巴胺受体的细胞模型;TR-FRET cAMP方法检测结果显示所构建的多巴胺受体细胞模型可以反映化合物的激动和抑制作用。结论 所构建的DA受体表达细胞系可以通过TR-FRET cAMP方法检测多巴胺受体被激活后胞内产生的第二信使cAMP的浓度变化,进而反映化合物对DA靶点的活性影响,为DA靶点的化合物筛选提供一种简便快捷的方法。Objective A cell-level assay of cAMP,a second messenger,was established to evaluate the effect of the compound on the activity of Dopamine(DA)receptors.Methods The eukaryotic expression vector of DA receptor was constructed,and Chinese hamster ovarian(CHO)cells expressing DA receptor were constructed on Flp-In system by liposome transfection.The expression of DA receptor was identified by reverse transcription PCR(RT-PCR).TR-FRET cAMP method was used to detect the activation and inhibition of positive compounds on DA targets.Forskolin(Forskolin)is a potent Adenylate cyclase(AC)activator that induces intracellular cAMP formation.Results As identified by RT-PCR,No target gene was expressed in the host cells,but target gene was expressed in the transfected cell lines.The cell model expressing dopamine receptor was constructed successfully.TR-FRET cAMP method showed that the constructed dopamine receptor cell model could reflect the excitatory and inhibitory effects of the compound.Conclusion The constructed cell model can be used to detect the concentration of the second messenger cAMP in the cytoplasm by TR-FRET cAMP method to reflect the effect of the compound on the activity of DA target,which provides a simple and fast method for compound screening of DA target.
关 键 词:多巴胺受体 细胞模型 第二信使 cAMP浓度 化合物筛选
分 类 号:R329.2[医药卫生—人体解剖和组织胚胎学]
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