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mRNA的qRT-PCR。使用1％多聚甲醛将剩余的细胞交联10分钟，然后加入甘氨酸并将样品离心（4℃，4000rpm，5分钟）以停止进一步交联。使用染色质免疫沉淀测定试剂盒进行染色质免疫沉淀。通过实时PCR扩增免疫沉淀的DNA。引物是PSA增强子前向-ATGTTCACATTAGTACACCTTGCC和反向TCTCAGATCCAGGCTTGCTTACTGTC和TMPRSS2增强子前向-TGGTCCTGGATGATAAAAAAAGTTT和反向-GACATACGCCCCACAACAGA [1]。",{"key":238,"value":239},"动物实验","小鼠[3]在5天的适应期后，阉割5至9周龄的雄性CB17SCID小鼠，并在接种肿瘤细胞之前再恢复5天。共表达外源AR的LNCaP细胞和AR依赖性报道构建体ARR2-Pb-Luc（LNCaP-AR-Lux细胞）用于产生人前列腺癌的异种移植模型。植入前，通过加入胰蛋白酶-EDTA制备LNCaP-AR-Lux细胞，用完全培养基洗涤，收集并以20×10 6个细胞\u002F mL重悬。将细胞悬浮液用Matrigel稀释至2×10 6个细胞\u002F0.2mL，并在肩胛上区域皮下递送。当治疗开始（80天）时，监测肿瘤生长至100mm 3的体积。观察到LNCaP-AR-Lux细胞的肿瘤摄取率在70％至80％之间。用数字卡尺每周测量体重和肿瘤体积（宽度2×长度\u002F 2）两到三次，并测定平均肿瘤体积。将试验药物在吐温80：PEG 400中稀释，并在4℃下储存直至通过口服强饲法给药。每组小鼠（n = 7）每天用1,10或50mg \u002F kg恩杂鲁胺，载体对照或50mg \u002F kg比卡鲁胺治疗连续28天。在治疗期结束时或当肿瘤体积超过1,000mm 3时，对动物实施安乐死并收集血液和组织样品用于分析。大鼠[4]雄性SD大鼠（n = 3）通过尾静脉（静脉内）和通过1mg \u002F kg的口服强饲法施用恩杂鲁胺，并在给药后保持在代谢笼中。在给药后的以下时间间隔收集尿液和粪便样品：0-2,2-4,4-6,6-10,10-24,24-48和48-72小时。用蒸馏水冲洗代谢笼，并在72小时时将残留物加入尿液样品中。为了提取粪便中存在的恩杂鲁胺，将样品用50％甲醇剧烈摇动12小时。",{"key":241,"value":242},"参考文献","[1].\nTran C, et al. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science, 2009, 324 (5928), 787-790.\n[2].\nScher HI, et al. Antitumour activity of MDV3100 in castration-resistant prostate cancer: a phase 1-2 study. Lancet, 2010, 375(9724), 1437-1446.\n[3].\nGuerrero J, et al. Enzalutamide, an androgen receptor signaling inhibitor, induces tumor regression in a mouse model of castration-resistant prostate cancer. Prostate. 2013 Sep;73(12):1291-305.\n[4].\nKim TH, et al. Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats. 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