桂枝茯苓丸对子宫内膜异位症间质细胞之差异性蛋白质组学研究

Based on previous and our recent results, we hypothesize that Guizhi Fuling Wan have excellent therapeutic effects on endometriosis (Ems), and the potential mechanisms of action might be hierarchical and involved in multiple molecular targets; meanwhile, ectopic endometrial stromal cells (ESCs) might be one of the main participants in the pathogenesis of endometriosis and thus a potential therapeutic target. The present project will firstly transplant subcutaneously ESCs into nude mice with controlled physiological cycles, feed mice with Guizhi Fuling Wan via intragastric administration, determine its pharmacologically therapeutic effects; collect the drug sera of treated mice for further study. Then we will homogenate the xenograft and screen and characterize the differentially expressed proteins using proteomic technique, construct relevant MAP profiles; among those proteins with differential high-expression, select four representative proteins which would be the potentially responsible targets for the four components, Jun, Chen, Zuo and Shi within the Guizhi Fuling Wan, and designate as X1, X2, X3 and X4. Subsequently, construct corresponding transgenic cells with RNAi manipulation; treated these transgenic cells with above-mentioned drug sera and determine the effects on cell proliferation, migration, cell cycle and apoptosis after 48 hours. Finally, both the native and transgenic cells were transplanted into nude mice to confirm these effects, to decipher the potential mechanisms of molecular biology of the X1, X2, X3 and X4 proteins with high expression, with the aim of providing theoretical and experimental bases for enriching and improving the theory of traditional Chinese medicine.

在总结前人与自身工作基础上，我们假设：桂枝茯苓丸对子宫内膜异位症（Ems）确有很好疗效，其机制是多层次、多靶点的；异位子宫内膜间质细胞（ESCs）是主要原因之一，也很可能成为Ems治疗新靶点。拟将ESCs异位接种至人工控制生理周期裸鼠皮下，灌胃给予桂枝茯苓丸，确认已发挥疗效；取含药血清备用；再取移植物制备组织匀浆，采用蛋白质组学技术，筛选鉴定其差异表达蛋白，绘制相应MAP图；按桂枝茯苓丸方解中之君、臣、佐、使，分别选择一个典型的差异性高表达蛋白X1、X2、X3、X4，采用RNAi技术，建立相应的转基因细胞；将上述含药血清分别加入母本细胞及转基因细胞中，48 h后检测细胞增殖率、迁移率、细胞周期及凋亡情况；最后将母本细胞及转基因细胞，分别接种至整体裸鼠行回复验证实验，以求阐明差异性高表达的蛋白X1、X2、X3、X4在桂枝茯苓丸治疗Ems中的分子生物学机制，为充实和完善中医药理论提供理论依据。

子宫内膜异位症是子宫腔外其他部位存在子宫内膜细胞，如卵巢，输卵管，膀胱和子宫肌层等。主要病理变化是异位子宫内膜出血和周围组织纤维化。子宫内膜异位症的主要症状是异位结节，痛经，慢性盆腔疼痛，月经不调和不孕症的形成，严重影响年轻女性的健康和生活质量。由于其发病机制尚不清楚，因此仍是妇产科领域的一大难题。中医药历史悠久，有助于各种疾病的预防和治疗。桂枝茯灵丸（GZFLW）是中国古代中药的配方之一，由五种生药组成，包括肉桂皮，茯苓，牡丹皮，柏油精和芍药。虽然GZFLW最初用于治疗妇科疾病，但其对子宫内膜异位症的分子机制尚不清楚。在这里，我们分离并培养了原代异位子宫内膜间质细胞（EESCs，这里命名为Y14），加入含GZFLW的血清进行处理后提取蛋白，然后使用iTRAQ蛋白质组学方法，发现差异表达蛋白VPS53。我们的研究结果表明，GZFLW可以通过促进其稳定性来增加VPS53的表达，并通过线粒体诱导的凋亡途径诱导子宫内膜异位症间质细胞的凋亡。