新型选择性肿瘤坏死因子-α转化酶抑制剂的HPLC筛选方法的研究

发布时间：2018-01-07 08:09

  本文关键词：新型选择性肿瘤坏死因子-α转化酶抑制剂的HPLC筛选方法的研究　出处：《华中科技大学》2008年硕士论文　论文类型：学位论文

  更多相关文章： TACE抑制剂 HPLC 内标法 半数抑制浓度 MMP-9

【摘要】： 肿瘤坏死因子-α转化酶(TACE或ADAM17)属于含锌金属蛋白酶家族。目前大量的实验研究发现,TACE具有重要的生理和病理作用,其功能涉及到调节免疫系统、诱发炎症性肠病以及神经性疾病等。TACE能催化水解人体内的跨膜型肿瘤坏死因子-α(pro-TNF-α)转化为可溶性的sTNF-α;诱发炎症性疾病。因此,TACE成为治疗炎症性疾病的新靶标。 人工合成的小分子抑制剂通过抑制TACE活性,进而控制炎症时人体内过高的sTNF-α水平,因此,寻找这些小分子抑制剂变的尤为重要,而建立有效评价酶抑制剂活性的方法是筛选这些小分子抑制剂的关键。本文就致力于建立评价TACE抑制剂的活性的方法。具体研究内容如下: 1.采用HPLC法评价TACE抑制剂活性。依据底物(12肽)的结构,合成了一种新化合物:DL-α-氨基丙酸- Dpa,作为定量分析的内标物。对酶浓度、TACE酶促反应时间、流动相等实验条件进行了优化,获得最佳测试条件,建立了使用内标法定量测定TACE活性的HPLC法;其线性范围为10～400μmol/L,检出限(LOD)为0.1μmol/L。然后,在最佳实验条件下,用此法测定抑制剂GM6001抑制TACE的半数抑制浓度IC50值。最后,用此新方法测定标准样品,其日内和日间的RSD都分别小于5%和10%;回收率在85%～115%间。 2. HPLC法评价MMPs抑制剂活性。为了满足选择性TACE抑制剂筛选的需要,还必须要建立评价MMPs抑制剂的方法。本实验以MMP-9作为MMPs家族的代表酶,选用连有紫外基团的七肽作为底物,优化酶促反应的实验条件,获得最佳测试条件,建立了使用内标法定量测定MMP-9活性的HPLC法;其线性范围为10～360μmol/L,检出限(LOD)为0.05μmol/L。然后,用此法评价了抑制剂GM6001对MMP-9的抑制作用,其结果与文献报道的结果基本一致。
[Abstract]:The tumor necrosis factor - 伪 - converting enzyme ( TACE ) is a family of zinc - containing metal proteases . A large number of experimental studies have found that TACE has important physiological and pathological effects . Its function involves regulating the immune system , inducing inflammatory bowel disease and neurological diseases . TACE can catalyze the transformation of transmembrane type tumor necrosis factor - 伪 ( pro - TNF - 伪 ) into soluble sTNF - 伪 in human body . TACE can be used as a new target for the treatment of inflammatory diseases . Artificial synthesis of small molecule inhibitors can control the activity of TACE and then control the levels of sTNF - . alpha . in human body during inflammation . Therefore , it is important to find these small molecule inhibitors , and the establishment of effective methods for evaluating the activity of enzyme inhibitor is the key to screening these small molecule inhibitors . The present paper is devoted to the establishment of methods for evaluating the activity of TACE inhibitors . 1 . The activity of TACE inhibitors was evaluated by HPLC . A new compound : DL - 伪 - aminopropionic acid - Dpa was synthesized according to the structure of substrate ( 12 - peptide ) . An HPLC method was established for the determination of TACE activity using internal standard . The linear range was 10 - 400 渭mol / L and the detection limit ( LOD ) was 0.1 渭mol / L . The standard samples were determined by this new method . The RSD was less than 5 % and 10 % , respectively , and the recovery was 85 % 锝，

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