目的 分別構(gòu)建穩(wěn)定過表達胰島素受體（IR） A、IRB蛋白的犬腎細胞MDCK，用于胰島素受體結(jié)合能力體外檢測。方法 利用慢病毒感染法在MDCK細胞內(nèi)分別穩(wěn)定過表達IRA、IRB蛋白，構(gòu)建MDCK-IRA、MDCK-IRB細胞，同時制備感染空白對照質(zhì)粒的MDCK-mock細胞，Western blotting檢測IR蛋白表達，¹²⁵IPCR（qRT-PCR）法檢測IR、IRB mRNA水平。進行¹²⁵I標記胰島素（1 nmol·L^-1）與MDCK-IRA、MDCK-IRB細胞結(jié)合動力學實驗，篩選孵育溫度、時間、細胞接種數(shù)；以中國食品藥品檢定研究院（中檢院）生物甘精胰島素粉末與來得時公司生產(chǎn)的甘精胰島素注射液作為胰島素受試品，進行與¹²⁵I標記胰島素的競爭結(jié)合實驗。結(jié)果 與MDCK-mock細胞比較，MDCK-IRA與MDCK-IRB細胞均高表達IR蛋白； MDCK-IRA與MDCK-IRB細胞均檢測出轉(zhuǎn)錄較高水平的IR mRNA，但IRB特異引物只能在MDCK-IRB細胞中檢測到IRB mRNA，均顯著升高（P＜0.01、0.001）。2種細胞模型具有胰島素結(jié)合作用，MDCK-IRA與胰島素的結(jié)合能力強于MDCK-IRB細胞，¹²⁵I標記胰島素與MDCK-IRA、MDCK-IRB細胞最佳孵育溫度與時間為4℃條件下孵育10 h，最適接種細胞數(shù)量為每孔5× 10⁵個細胞以內(nèi)。不同濃度來得時和中檢院甘精胰島素對¹²⁵I標記胰島素與IRA結(jié)合活性抑制作用的半數(shù)抑制濃度（IC₅₀）值分別為3.698、5.829 nmol·L^-1，對¹²⁵I標記胰島素與IRB結(jié)合活性抑制作用IC₅₀值分別為4.977、9.068 nmol·L^-1。結(jié)論 過表達IRA、IRB蛋白的MDCK細胞模型簡便、功能穩(wěn)定，可用于體外檢測胰島素制劑與IR結(jié)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

藥物成藥性評價與系統(tǒng)轉(zhuǎn)化全國重點實驗室項目（712023001，712023002，712024001）