本文選題：Na~+依賴性葡萄糖攝取 + 鈉-葡萄糖協(xié)同轉(zhuǎn)運(yùn)蛋白��； 參考：《藥學(xué)學(xué)報(bào)》2017年06期

【摘要】：本研究的目的是建立SGLT2選擇性抑制劑篩選及藥效學(xué)評價(jià)體系。分別將人全長SGLT2和SGLT1c DNA連接至p MSCVpuro載體并轉(zhuǎn)染HEK293細(xì)胞,嘌呤霉素篩選單克隆細(xì)胞,RT-PCR、Western blot或細(xì)胞免疫熒光染色檢測目的基因、蛋白在細(xì)胞中的表達(dá)。采用熒光標(biāo)記的1-脫氧葡萄糖(1-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-1-deoxy-D-glucose,1-NBDG)作為底物進(jìn)行葡萄糖轉(zhuǎn)運(yùn)實(shí)驗(yàn),檢測模型細(xì)胞的Na+依賴的葡萄糖攝取功能,并對陽性藥達(dá)格列凈、根皮苷選擇性進(jìn)行評價(jià)。最后,進(jìn)行整體藥效評價(jià),觀察達(dá)格列凈對正常小鼠及四氧嘧啶誘導(dǎo)的T1DM小鼠血糖的影響。結(jié)果顯示p MSCVpuro-SGLT1轉(zhuǎn)染組細(xì)胞SGLT1基因及蛋白水平均顯著升高,p MSCVpuro-SGLT2轉(zhuǎn)染組SGLT2蛋白高表達(dá)于細(xì)胞膜及細(xì)胞質(zhì),說明SGLT1/SGLT2穩(wěn)定過表達(dá)細(xì)胞構(gòu)建成功。葡萄糖轉(zhuǎn)運(yùn)實(shí)驗(yàn)結(jié)果顯示,與p MSCVpuro-null轉(zhuǎn)染組相比,p MSCVpuro-SGLT1、p MSCVpuroSGLT2轉(zhuǎn)染組細(xì)胞Na~+依賴性1-NBDG攝取明顯增高。達(dá)格列凈具有良好的選擇性,對SGLT1的半數(shù)抑制濃度(IC50,6.20×10~(-7) mol·L~(-1))遠(yuǎn)高于對SGLT2抑制的IC50值(2.24×10~(-10) mol·L~(-1)),優(yōu)于非選擇性抑制劑根皮苷。達(dá)格列凈單次給藥即可改善正常小鼠葡萄糖耐量。對T1DM小鼠,達(dá)格列凈從給藥后1 h血糖水平即顯著降低,能夠劑量依賴地降低0~24 h血糖-時(shí)間曲線下面積。連續(xù)給藥20天,能夠較平穩(wěn)地降低小鼠餐后血糖。本研究建立了較完善的SGLT2選擇性抑制劑的體外篩選方法和整體藥效學(xué)評價(jià)方法。該方法具有無同位素污染,高轉(zhuǎn)運(yùn)效率、良好穩(wěn)定性等優(yōu)點(diǎn),將為新的SGLT2高選擇性抑制劑研發(fā)提供良好的技術(shù)平臺。
[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.
【作者單位】： 中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院藥物研究所新藥作用機(jī)制研究與藥效評價(jià)北京市重點(diǎn)實(shí)驗(yàn)室天然藥物活性物質(zhì)與功能國家重點(diǎn)實(shí)驗(yàn)室;
【基金】：中國醫(yī)學(xué)科學(xué)院醫(yī)學(xué)與健康科技創(chuàng)新工程團(tuán)隊(duì)項(xiàng)目(CIFMS-2016-I2M-3-012) 天然藥物活性物質(zhì)與功能國家重點(diǎn)實(shí)驗(yàn)室開放基金(GTZK201512)
【分類號】：R96

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