目的 分别构建稳定过表达胰岛素受体（IR） A、IRB蛋白的犬肾细胞MDCK，用于胰岛素受体结合能力体外检测。方法 利用慢病毒感染法在MDCK细胞内分别稳定过表达IRA、IRB蛋白，构建MDCK-IRA、MDCK-IRB细胞，同时制备感染空白对照质粒的MDCK-mock细胞，Western blotting检测IR蛋白表达，¹²⁵IPCR（qRT-PCR）法检测IR、IRB mRNA水平。进行¹²⁵I标记胰岛素（1 nmol·L^-1）与MDCK-IRA、MDCK-IRB细胞结合动力学实验，筛选孵育温度、时间、细胞接种数；以中国食品药品检定研究院（中检院）生物甘精胰岛素粉末与来得时公司生产的甘精胰岛素注射液作为胰岛素受试品，进行与¹²⁵I标记胰岛素的竞争结合实验。结果 与MDCK-mock细胞比较，MDCK-IRA与MDCK-IRB细胞均高表达IR蛋白； MDCK-IRA与MDCK-IRB细胞均检测出转录较高水平的IR mRNA，但IRB特异引物只能在MDCK-IRB细胞中检测到IRB mRNA，均显著升高（P＜0.01、0.001）。2种细胞模型具有胰岛素结合作用，MDCK-IRA与胰岛素的结合能力强于MDCK-IRB细胞，¹²⁵I标记胰岛素与MDCK-IRA、MDCK-IRB细胞最佳孵育温度与时间为4℃条件下孵育10 h，最适接种细胞数量为每孔5× 10⁵个细胞以内。不同浓度来得时和中检院甘精胰岛素对¹²⁵I标记胰岛素与IRA结合活性抑制作用的半数抑制浓度（IC₅₀）值分别为3.698、5.829 nmol·L^-1，对¹²⁵I标记胰岛素与IRB结合活性抑制作用IC₅₀值分别为4.977、9.068 nmol·L^-1。结论 过表达IRA、IRB蛋白的MDCK细胞模型简便、功能稳定，可用于体外检测胰岛素制剂与IR结合活性。

Objective To construct canine kidney cells MDCK stably overexpressing insulin receptor (IR) A and IRB proteins for in vitro detection of insulin receptor binding ability. Methods MDCK cells were stably overexpressed with IRA and IRB proteins by lentivirus infection to construct MDCK-IRA and MDCK-IRB cells, and MDCK-mock cells infected with blank control plasmid were prepared. Western blotting was used to detect IR protein expression, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect IR and IRB mRNA levels. The binding kinetics experiments of ¹²⁵I-labeled insulin (1 nmol·L^-1) with MDCK-IRA and MDCKIRB cells were conducted to screen the incubation temperature, time, and cell seeding number. The insulin products of biosimilar insulin glargine powder from the National Institute for Food and Drug Control and insulin glargine injection from Sanofi-Aventis were used as test samples for competitive binding experiments with ¹²⁵I-labeled insulin (1 nmol·L^-1). Results Compared with MDCKmock cells, both MDCK-IRA and MDCK-IRB cells highly expressed IR protein. Both MDCK-IRA and MDCK-IRB cells showed high transcription levels of IR mRNA, but IRB-specific primers could only detect IRB mRNA in MDCK-IRB cells, which were significantly increased (P < 0.01, 0.001). The two cell models had similar insulin binding effects, but the binding ability of MDCK-IRA to insulin was slightly stronger than that of MDCK-IRB cells. The optimal incubation temperature and time for ¹²⁵I-labeled insulin with MDCKIRA and MDCK-IRB cells were 4 ℃ for 10 h, and the optimal cell seeding number was less than 5×10⁵ cells per well. The halfmaximal inhibitory concentration (IC₅₀) values of different concentrations of biosimilar insulin glargine powder from the National Institute for Food and Drug Control and insulin glargine injection from Sanofi-Aventis on the binding activity of ¹²⁵I-labeled insulin to IRA were 3.698 and 5.829 nmol·L^-1, respectively, and the IC₅₀ values on the binding activity of ¹²⁵I-labeled insulin to IRB were 4.977 and 9.068 nmol·L^-1, respectively. Conclusion The MDCK cell models overexpressing IRA and IRB proteins are simple and functionally stable, and can be used for in vitro detection of the binding activity of insulin preparations to IR.

R965

药物成药性评价与系统转化全国重点实验室项目（712023001，712023002，712024001）