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，将上清液保存在-80℃。通过将20μL适当的化合物稀释液与50μL用于DPP-4酶，H-Ala-Pro-7-酰氨基-4-三氟甲基香豆素（测定中的最终浓度，100μM）和30μL的底物混合来进行测定。 Caco-2细胞提取物（用100mM Tris-HCl，100mM NaCl，pH7.8稀释1000倍）。将板在室温下孵育1小时，并使用SpectraMax GeminiXS在405 \u002F 535nm的激发\u002F发射波长下测量荧光。在具有高抑制剂浓度的Caco-2细胞提取物预孵育1小时后测定来自DPP-4酶的抑制剂的解离动力学（BI 1356为30nM，维达列汀为3μM）。在用测定缓冲液稀释预孵育混合物3000倍后，通过加入底物H-Ala-Pro-7-酰氨基-4-三氟甲基香豆素开始酶促反应。在这些条件下，在存在或不存在抑制剂的情况下在某一时间点DPP-4活性的差异反映了仍然与DPP-4酶结合的该抑制剂的量。使用SpectraMax的SoftMax软件计算10分钟间隔的最大反应速率（荧光单位\u002F秒×1000），并校正未抑制反应的速率[（vcontrol-vinhibitor）\u002F vcontrol]。",{"key":250,"value":251},"细胞实验","将CD4T细胞接种在无血清RPMI 1640的膜插入物上，并在存在或不存在纯化的猪肾DPP-4（32.1单位\u002F mg; 100mU \u002F mL终浓度）的情况下使用Transwell小室（Corning）测定细胞迁移。浓度）和DPP-4抑制剂（100μM）。 1小时后，机械除去上表面的细胞，计数迁移到下室的细胞。迁移程度相对于对照样品表示。",{"key":253,"value":254},"动物实验","小鼠：在口服葡萄糖负荷（2g \u002F kg）的化合物给药后45分钟，对隔夜禁食的C57BL \u002F 6J小鼠进行攻击。用于葡萄糖测量的血液样品通过尾部给药前和葡萄糖负荷后的连续时间点获得。为了评估对葡萄糖耐量的影响持续时间，在葡萄糖激发前16小时施用载体或DPP-4抑制剂。",{"key":256,"value":257},"参考文献","[1].\nThomas, L., et al. (R)-8-(3-amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione (BI 1356), a novel xanthine-based dipeptidyl peptidase 4 inhibitor, has a superior potency and longer duration of action compared with other dipeptidyl peptidase-4 inhibitors. J Pharmacol Exp Ther. 2008 Apr;325(1):175-82.\n[2].\nKim, S.J., et al., Dipeptidyl peptidase IV inhibition with MK0431 improves islet graft survival in diabetic NOD mice partially via T-cell modulation. Diabetes, 2009. 58(3): p. 641-51.\n[3].\nSangle, G.V., et al., Novel biological action of the dipeptidylpeptidase-IV inhibitor, sitagliptin, as a glucagon-like peptide-1 secretagogue. Endocrinology, 2012. 153(2): p. 564-73.\n[4].\nKim, S.J., et al., Inhibition of dipeptidyl peptidase IV with sitagliptin (MK0431) prolongs islet graft survival in streptozotocin-induced diabetic mice. Diabetes, 2008. 57(5): p. 1331-9.\n[5].\nBeconi, M.G., et al. Disposition of the dipeptidyl peptidase 4 inhibitor sitagliptin in rats and dogs. 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