丹参酮IIA对子宫腺肌病异位间质细胞之差异蛋白质组学研究

Based on previous clinical experience of the treatment on adenomyosis(AM) with Chinese medicine including danshen and our recent results, we found Tanshinone IIA (TSIIA) weakened EESCs ability of survival, migration and invasion, and induce its apoptosis, so we hypothesize that Tanshinone IIA (TSIIA) have excellent therapeutic effects on adenomyosis (AM), and the potential mechanisms of action might be complex; meanwhile, ectopic endometrial stromal cells (EESCs) might　be a potential therapeutic target. In early , The present project will firstly combine 2 - D DIGE electrophoresis/MALDI - TOF - MS analysis technology, screen and characterize the differentially expressed proteins when TSIIA have curative effects in EESCs, then construct relevant MAP profiles; among those proteins with differential high-expression, select one representative proteins which would be the potentially responsible targets, and designate as X. Subsequently, construct corresponding transgenic cells with RNAi manipulation; treated these transgenic cells with TSIIA 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 X proteins with high expression in the treatment of adenomyosis (AM) by Tanshinone IIA (TSIIA), with the aim of providing theoretical and experimental bases for improving and enriching the theory of traditional Chinese medicine.

根据前人使用含有丹参的活血化瘀中药治疗AM的良好临床基础，总结自身前期实验基础上，我们发现丹参酮IIA能够减弱子宫腺肌病异位间质细胞（EESCs）的生存能力、迁移和侵袭能力，并诱导其凋亡。因此假设：丹参酮IIA对AM确有很好的疗效，且机制复杂；子宫腺肌病异位间质细胞（EESCs）有可能成为治疗AM的新靶点。拟将2-D DIGE电泳/MALDI-TOF-MS分析等技术有机结合，探讨丹参酮IIA对EESCs产生疗效时蛋白组学的差异性，绘制相应MAP图；选择一个典型的差异性高表达蛋白X，采用RNAi技术，建立相应的转基因细胞株；将丹参酮IIA分别加入母本细胞及转基因细胞中，48h后检测其细胞增殖率、迁移率、细胞周期以及凋亡情况；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X在丹参酮IIA治疗AM中的分子生物学机制，为完善和丰富中医药理论提供实验基础。

根据前人使用含有丹参的活血化瘀中药治疗AM的良好临床基础，总结自身前期实验基础上，我们发现丹参酮IIA能够减弱子宫腺肌病异位间质细胞（EESCs）的生存能力、迁移和侵袭能力，并诱导其凋亡。本课题结合前期研究结果后，采用蛋白质组学技术，探讨丹参酮IIA 对EESCs 产生疗效时蛋白组学的差异性。本课题通过iTRAQ标记定量蛋白质组学技术进行差异蛋白质组学鉴定，对于筛选出的差异高表达蛋白，通过生信分析及文献搜索，我们遴选了两个个典型的候选差异蛋白TNFRSF10D以及GDF15。通过进一步的细胞功能学验证，证明这些蛋白在丹参酮IIA抑制子宫腺肌症原代细胞中增殖和促进细胞凋亡的过程中无显著性作用。目前对于丹参酮IIA抑制间质细胞的关键蛋白仍在探寻当中。同时，在本课题的资助下，课题组成员成员成功构建了裸鼠皮下移植的异位病灶，经HE染色，免疫荧光鉴定，该病灶组织与人异位病灶相似。以上的结果对挖掘丹参酮IIA治疗AM可能作用的效应/靶蛋白，以及为完善和丰富中医药理论提供实验理论依据。