SILAC/AACT结合质谱MRM技术解析苦参碱及其改构衍生物YYJ18对鼻咽癌细胞抑制作用机制

NPC is one of the high prevalence of malignant tumors in Guangxi. Clinical practice found matrine with anti-nasopharyngeal carcinoma effect, but its mechanism was unknown and the anti-tumor effect was limited. Our study found that derivative YYJ18 from matrine(that has been applied for a patent)had more specific inhibitory effect in the growth of NPC at the prophase. At the same time cytological experiments found that the effect associated with some important protein signaling pathway. By intending to use SILAC/AACT that based on mass spectrometry(MS),we analyse quantificationally the changes of nasopharyngeal carcinoma cell in the proteomics of different cellular components causing by matrine and its derivative JYY18 ,especially comparing differences in protein network changed by matrine and its derivative JYY18;then we verify a large number of differentially expressed proteins which have been screened by mass spectrometry and MRM approach;and we analyse synthetically the discoverable differential proteins in bioinformatics and select important proteins to study their function.Finally,we build a series of key proteins to a protein chip,investigate the mode of action of the other matrine derivatives. This work will provide theoretical and experimental support for clarifying the acting mechanism of matrine drugs and its modification derivatives and for the screen and use of drug modification, also provide a useful therapeutic strategy for the clinical treatment of nasopharyngeal carcinoma.

鼻咽癌是广西高发的恶性肿瘤之一。临床发现苦参碱具有抗鼻咽癌效果，但其机理不明、作用有限。本课题组前期发现以苦参碱改构获得的衍生物YYJ18具有更强的特异性抑制鼻咽癌生长的效果；同时细胞学实验发现，该效果与一些重要的蛋白信号通路相关。本项目拟利用以质谱为基础的稳定同位素氨基酸定量标记技术（SILAC/AACT），定量分析苦参碱及其改构衍生物YYJ18引起的鼻咽癌细胞在不同细胞组分蛋白质组学的改变，特别比较苦参碱与YYJ18引起的蛋白网络改变的异同；然后将筛选到的大量差异表达蛋白通过质谱MRM技术进行验证；将找到的差异蛋白进行生物信息学综合分析，选取重要蛋白研究其功能；最后将关键的一系列蛋白构建蛋白芯片，考察其它苦参碱改构衍生物的作用方式。该工作将为阐明苦参碱类药物及其改构衍生物的作用机理，为我们以后改构药物的筛选及应用提供理论及实验支持；也为鼻咽癌的临床治疗提供有益的治疗策略。

鼻咽癌是广西常见的头颈部恶性肿瘤之一。 临床上发现苦参碱具有抗鼻咽癌作用，但其效率太低，我们通过对苦参碱进行改构获得苦参碱衍生物，具有更强的特异性抑制鼻咽癌生长。课题利用以质谱为基础的稳定同位素氨基酸定量标记技术（SILAC/AACT），定量分析苦参碱及其改构衍生物YYJ18引起的鼻咽癌细胞（CNE2）在不同细胞组分蛋白质组学的改变，比较苦参碱与YYJ18引起的蛋白网络改变的异同；然后将筛选到的大量差异表达蛋白通过质谱MRM技术、荧光定量PCR及免疫蛋白印迹技术进行验证，以确定差异蛋白。结果显示：（1）苦参碱及苦参碱衍生物能有效抑制鼻咽癌细胞CNE2的生长，（2）苦参碱及苦参碱衍生物通过上调和下调相关蛋白发挥抗鼻咽癌作用。本课题为我们阐明了苦参碱及其衍生物通过上调和下调相关蛋白能有效诱导鼻咽癌细胞凋亡。