SGLT2选择性抑制剂筛选及药效评价方法建立

发布时间：2018-07-04 17:14

本文选题：Na~+依赖性葡萄糖摄取 + 钠-葡萄糖协同转运蛋白　；参考：《药学学报》2017年06期

【摘要】：本研究的目的是建立SGLT2选择性抑制剂筛选及药效学评价体系。分别将人全长SGLT2和SGLT1c DNA连接至p MSCVpuro载体并转染HEK293细胞,嘌呤霉素筛选单克隆细胞,RT-PCR、Western blot或细胞免疫荧光染色检测目的基因、蛋白在细胞中的表达。采用荧光标记的1-脱氧葡萄糖(1-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-1-deoxy-D-glucose,1-NBDG)作为底物进行葡萄糖转运实验,检测模型细胞的Na+依赖的葡萄糖摄取功能,并对阳性药达格列净、根皮苷选择性进行评价。最后,进行整体药效评价,观察达格列净对正常小鼠及四氧嘧啶诱导的T1DM小鼠血糖的影响。结果显示p MSCVpuro-SGLT1转染组细胞SGLT1基因及蛋白水平均显著升高,p MSCVpuro-SGLT2转染组SGLT2蛋白高表达于细胞膜及细胞质,说明SGLT1/SGLT2稳定过表达细胞构建成功。葡萄糖转运实验结果显示,与p MSCVpuro-null转染组相比,p MSCVpuro-SGLT1、p MSCVpuroSGLT2转染组细胞Na~+依赖性1-NBDG摄取明显增高。达格列净具有良好的选择性,对SGLT1的半数抑制浓度(IC50,6.20×10~(-7) mol·L~(-1))远高于对SGLT2抑制的IC50值(2.24×10~(-10) mol·L~(-1)),优于非选择性抑制剂根皮苷。达格列净单次给药即可改善正常小鼠葡萄糖耐量。对T1DM小鼠,达格列净从给药后1 h血糖水平即显著降低,能够剂量依赖地降低0~24 h血糖-时间曲线下面积。连续给药20天,能够较平稳地降低小鼠餐后血糖。本研究建立了较完善的SGLT2选择性抑制剂的体外筛选方法和整体药效学评价方法。该方法具有无同位素污染,高转运效率、良好稳定性等优点,将为新的SGLT2高选择性抑制剂研发提供良好的技术平台。
[Abstract]:The aim of this study was to establish a selective inhibitor screening and pharmacodynamics evaluation system for SGLT2. Human full-length SGLT2 and SGLT1c DNA were ligated into pMSCVpuro vector and transfected into HEK293 cell line respectively. Purine mycin was used to screen monoclonal cell line RT-PCRN Western blot or cellular immunofluorescence staining to detect the expression of target gene and protein in HEK293 cells. Glucose transport experiments were carried out by using fluorescence labeled 1- [N- (7-nitrobenz-2-oxa-1oxa-1-diazol-4-yl) amino] -1-deoxy-D-glucose 1-NBDG as substrate to detect the Na dependent glucose uptake function of model cells. Finally, the effect of daglidine on blood glucose in normal mice and alloxan induced T1DM mice was evaluated. The results showed that the levels of SGLT1 gene and protein in pMSCVpuro-SGLT1 transfected group were significantly higher than those in pMSCVpuro-SGLT2 transfection group, which indicated that SGLT1 / SGLT2 stably overexpressed cells were successfully constructed in pMSCVpuro-SGLT2 transfection group. Compared with the pMSCVpuro-SGLT1 transfection group, the uptake of 1-NBDG in the pMSCVpuro-SGLT1 pMSCVpuroSGLT2 transfected cells was significantly higher than that in the pMSCVpuro-null transfection group. The half inhibitory concentration (IC50 ~ (-20) 脳 10 ~ (-7) mol L ~ (-1) for SGLT1 was much higher than that for SGLT2 (2.24 脳 10 ~ (-10) mol L ~ (-1),). A single dose of daglidine can improve glucose tolerance in normal mice. For T1DM mice, the level of blood glucose decreased significantly from 1 hour after administration of daglidine, and the area under the blood glucose-time curve of 0 ~ 24 h was decreased in a dose-dependent manner. After continuous administration for 20 days, the postprandial blood glucose of mice could be reduced steadily. In this study, we established a better method for screening SGLT2 selective inhibitors in vitro and evaluating their overall pharmacodynamics. This method has the advantages of no isotope pollution, high transport efficiency and good stability. It will provide a good technical platform for the development of new SGLT2 high selectivity inhibitors.
【作者单位】：中国医学科学院北京协和医学院药物研究所新药作用机制研究与药效评价北京市重点实验室天然药物活性物质与功能国家重点实验室;
【基金】：中国医学科学院医学与健康科技创新工程团队项目(CIFMS-2016-I2M-3-012) 天然药物活性物质与功能国家重点实验室开放基金(GTZK201512)
【分类号】：R96

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