基于iTRAQ技术结合2D―LC―MSMS研究黄连对CYP450同工酶表达影响.docVIP

基于iTRAQ技术结合2D―LC―MSMS研究黄连对CYP450同工酶表达影响 　　[摘要]黄连是临床常用药材，在中医药中被广泛应用，具有清热燥湿、泻火解毒之功，中药成分比较复杂，细胞色素P450（cytochrome P450， CYP450）同工酶是药物的主要代谢酶，研究黄连对CYP450酶的影响具有很重要的临床意义。实验选用大鼠肝脏组织，差速离心法分离肝微粒体，BCA（bicinchoninic acid）蛋白定量，应用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantification， iTRAQ）技术结合 2D-LC-MS/MS筛选差异蛋白。黄连一共鉴定出30个CYP450 同工酶，7个表达上调，8个表达下调，15个没有发生变化。iTRAQ技术结合2D-LC-MS/MS可以全面研究CYP450酶，但黄连有必要进一步在体外水平对其进行评价，预防潜在的临床药物相互作用。 　　[关键词]黄连；同位素标记相对和绝对定量技术（iTRAQ）；CYP45同工酶；2D-LC-MS/MS；蛋白质组学 　　[Abstract]Coptidis Rhizoma is commonly used in clinical medicine. It has been widely used in traditional Chinese medicine （TCM）， with the functions of clearing heat， drying dampness， purging intense heat relieve toxins. However， the herb contains complex ingredients， and CYP450 isoenzyme is the main metabolic enzyme of the drug， so it is of important clinical significance to study the effect of Coptidis Rhizoma on cytochrome P450 enzymes. In the experiment， liver tissues of rats were selected and liver microsomes were separated by differential centrifugation. Bicinchoninic acid （BCA） Protein quantitation was done by enzyme linked immunosorbent assay， and iTRAQ （isobaric tags for relative and absolute quantification） was combined with the 2D-LC-MS/MS analysis method for identification of CYP450 proteins. With Coptidis Rhizoma， 30 CYP450 isoenzymes were identified， with 7 proteins significantly increased and 8 decreased in response to Coptidis Rhizoma， while the rest 15 had no change. iTRAQ technology combined with 2D-LC-MS/MS method could be used to comprehensively study CYP450 enzyme， but it is necessary to further evaluate in vitro levels of Coptidis Rhizoma and avoid any potential clinical drug-drug interaction. 　　[Key words]Coptidis Rhizoma； iTRAQ； CYP450； 2D-LC-MS/MS； proteomics 　　黄连Coptidis Rhizoma是毛茛科植物、三角叶或云连的干燥根茎，中国传统草药，味苦、性寒，入心、脾、胃、肝、胆囊、大肠经。具清热燥湿、泻火解毒之功。黄连主要含有生物碱、小檗碱、黄连碱、黄连宁、巴马汀等化学成分。现在药理学研究表明：黄连具有降血糖、抗癌、免疫调节、改善心血管、降血压等药效，其中降低血糖、调节心血管功能和抗癌作用是近年来黄连药理研究的重点[1]。黄连作为清热解毒中药，不仅药