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蛋白\n>>\nLPL受体\n研究领域\n>>\n炎症\u002F免疫",{"key":239,"value":240},"靶点","IC50: 0.98 μM (human LPA1), 0.73 μM (mouse LPA1)",{"key":242,"value":243},"体外研究","AM095抑制用人或小鼠LPA1稳定转染的CHO细胞的LPA诱导的钙通量。 AM095拮抗LPA诱导的人或小鼠LPA1转染的CHO细胞的钙通量的IC50分别为0.025和0.023μM[1]。与载体对照相比,AM095在10μM时将LPA诱导的血管舒张减少约90％[2]。 AM095抑制LPA驱动的过表达小鼠LPA1(IC50 = 778 nM)和人A2058黑素瘤细胞(IC50 = 233 nM)的CHO细胞的趋化性[3]。",{"key":245,"value":246},"体内研究","LPA1与AM095的药理学拮抗作用显着减弱博来霉素诱导的皮肤纤维化[1]。 AM095具有高口服生物利用度和中等半衰期,并且在口服和静脉内给药后在大鼠和狗中测试的剂量下具有良好的耐受性。 AM095剂量依赖性地降低LPA刺激的组胺释放。 AM095减弱博莱霉素诱导的支气管肺泡灌洗液中胶原蛋白,蛋白质和炎性细胞浸润的增加。 AM095在小鼠单侧输尿管梗阻模型中减少肾纤维化[3]。",{"key":248,"value":249},"激酶实验","使用hLPA1 \u002F CHO和mLPA1 \u002F CHO细胞进行测定。将hLPA1 \u002F CHO或mLPA1 \u002F CHO细胞的细胞沉淀重悬于20mL含有10mM HEPES，pH7.4,1mM二硫苏糖醇和蛋白酶抑制剂的冰冷的膜缓冲液中。对细胞进行超声处理，并将细胞裂解物在4℃下以2000rpm离心10分钟。将上清液在4℃以25,000rpm进一步离心70分钟。将膜沉淀重悬于5mL冰冷的膜缓冲液中，并使用Potter-Elvehjem组织研磨机均化。使用Bradford Protein Assay Kit测定最终蛋白质浓度。将已知量的AM095（在二甲基亚砜中稀释）或载体（二甲基亚砜）加入25至40μg的hLPA1 \u002F CHO或mLPA1 \u002F CHO膜和0.1nM [35S]-GTPγS的缓冲液（50mM HEPES，0.1mM NaCl）中。 ，含有0.2％不含脂肪酸的人血清白蛋白和5μMGDP的10mMMgCl2,50μg\u002F mL皂苷，pH7.5）。为了测试LPA1拮抗剂活性，测量AM095抑制由900nM LPA（18：1）刺激的GTPγS结合的能力。或者，为了测试激动剂作用，测量AM095在不存在LPA的情况下刺激GTPγS结合的能力。将反应物在30℃温育30分钟，然后将膜收获到玻璃滤膜结合板上，并使用Brandel 96-尖端细胞收集器用含有50mM HEPES，pH 7.4,100mM NaCl，10mM MgCl 2的冷缓冲液洗涤三次。将板干燥，然后使用Packard TopCount NXT微孔板闪烁计数器评估cpm。",{"key":251,"value":252},"动物实验","小鼠对左肾进行UUO或假手术。简言之，进行纵向左上切口以暴露左肾。定位肾动脉并在动脉和输尿管之间通过6\u002F0丝线。将线缠绕在输尿管周围并打结三次以确保输尿管完全结扎。将肾脏返回腹部，缝合腹部肌肉，并用钉子将皮肤闭合。对侧（右）肾脏作为未受伤的对照。在UUO之前1至4小时给予AM095（30mg \u002F kg）或载体（水），然后通过口服强饲法进行投标。 8天后，使用吸入的CO 2对小鼠实施安乐死，收获肾脏并切成两半用于纤维化的组织病理学和生化分析。为了评估纤维化，将一半肾脏固定在10％中性缓冲福尔马林中并使用Masson三色染色。将另一半肾脏在-80℃冷冻，用于随后的胶原蛋白含量的生化分析。",{"key":254,"value":255},"参考文献","[1].\nCastelino FV, et al. Amelioration of dermal fibrosis by genetic deletion or pharmacologic antagonism of lysophosphatidic acid receptor 1 in a mouse model of scleroderma. Arthritis Rheum. 2011 May;63(5):1405-15.\n[2].\nRuisanchez E, et al. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase. FASEB J. 2014 Feb;28(2):880-90.\n[3].\nSwaney, J. S., et al. Pharmacokinetic and pharmacodynamic characterization of an oral lysophosphatidic acid type 1 receptor-selective antagonist. 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