發(fā)布時間：2018-01-07 08:09

  本文關(guān)鍵詞：新型選擇性腫瘤壞死因子-α轉(zhuǎn)化酶抑制劑的HPLC篩選方法的研究　出處：《華中科技大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： TACE抑制劑 HPLC 內(nèi)標(biāo)法 半數(shù)抑制濃度 MMP-9

【摘要】： 腫瘤壞死因子-α轉(zhuǎn)化酶(TACE或ADAM17)屬于含鋅金屬蛋白酶家族。目前大量的實驗研究發(fā)現(xiàn),TACE具有重要的生理和病理作用,其功能涉及到調(diào)節(jié)免疫系統(tǒng)、誘發(fā)炎癥性腸病以及神經(jīng)性疾病等。TACE能催化水解人體內(nèi)的跨膜型腫瘤壞死因子-α(pro-TNF-α)轉(zhuǎn)化為可溶性的sTNF-α;誘發(fā)炎癥性疾病。因此,TACE成為治療炎癥性疾病的新靶標(biāo)。 人工合成的小分子抑制劑通過抑制TACE活性,進而控制炎癥時人體內(nèi)過高的sTNF-α水平,因此,尋找這些小分子抑制劑變的尤為重要,而建立有效評價酶抑制劑活性的方法是篩選這些小分子抑制劑的關(guān)鍵。本文就致力于建立評價TACE抑制劑的活性的方法。具體研究內(nèi)容如下: 1.采用HPLC法評價TACE抑制劑活性。依據(jù)底物(12肽)的結(jié)構(gòu),合成了一種新化合物:DL-α-氨基丙酸- Dpa,作為定量分析的內(nèi)標(biāo)物。對酶濃度、TACE酶促反應(yīng)時間、流動相等實驗條件進行了優(yōu)化,獲得最佳測試條件,建立了使用內(nèi)標(biāo)法定量測定TACE活性的HPLC法;其線性范圍為10～400μmol/L,檢出限(LOD)為0.1μmol/L。然后,在最佳實驗條件下,用此法測定抑制劑GM6001抑制TACE的半數(shù)抑制濃度IC50值。最后,用此新方法測定標(biāo)準(zhǔn)樣品,其日內(nèi)和日間的RSD都分別小于5%和10%;回收率在85%～115%間。 2. HPLC法評價MMPs抑制劑活性。為了滿足選擇性TACE抑制劑篩選的需要,還必須要建立評價MMPs抑制劑的方法。本實驗以MMP-9作為MMPs家族的代表酶,選用連有紫外基團的七肽作為底物,優(yōu)化酶促反應(yīng)的實驗條件,獲得最佳測試條件,建立了使用內(nèi)標(biāo)法定量測定MMP-9活性的HPLC法;其線性范圍為10～360μmol/L,檢出限(LOD)為0.05μmol/L。然后,用此法評價了抑制劑GM6001對MMP-9的抑制作用,其結(jié)果與文獻報道的結(jié)果基本一致。
[Abstract]:The tumor necrosis factor - 偽 - 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 - 偽 ( pro - TNF - 偽 ) into soluble sTNF - 偽 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 - 偽 - 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，

本文編號：1391692