从Wnt/β-catenin通路探讨温肾消癥汤调控子宫内膜间充质干细胞分化的机制

Endometrial mesenchymal stem cells are closely associated with the development of endometriosis. The differentiation of endometrial mesenchymal stem cells into myofibroblasts is the biological basis of the pathological features of endometriosis. Blocking the pathway of endometrial mesenchymal stem cells differentiating into myofibroblasts and promoting endometrial mesenchymal stem cells differentiating into epithelial cells are new ideas for the treatment of endometriosis. Our previous studies have shown that the traditional Chinese medicine formula Wenshen Xiaozheng Tang, of which we have independent intellectual property rights, is effective in the treatment of endometriosis. The results of preliminary basic research showd that Wenshen Xiaozheng Tang inhibited endometrial mesenchymal stem cells differentiating into myofibroblasts while promoting them differentiating into epithelial cells. The underlying mechanism may relate to Wnt/β-catenin pathway. Therefore, we plan to study the association of Wnt/β-catenin pathway and the differentiation of endometrial mesenchymal stem cells regulated by Wenshen Xiaozheng Tang and to find the target of Wenshen Xiaozheng Tang in regulating Wnt/β-catenin signaling pathway both in vitro and in vivo using experimental techniques such as Western blot, qPCR, immunofluorescence and cell transfection. The results will provide the scientific basis for the clinical use of Wenshen Xiaozheng Tang in treating endometriosis and provide a theoretical basis to find new treatments for endoemtriosis in future.

子宫内膜间充质干细胞(MSCs)与子宫内膜异位症(EMs)的发生发展密切关联。子宫内膜MSCs分化为肌成纤维细胞是EMs病理特征的生物学基础。阻断子宫内膜MSCs向肌成纤维细胞分化的途径，促使其向上皮细胞分化，是治疗EMs新的思路和方法。前期研究表明，我院具有自主知识产权的中药复方温肾消癥汤在治疗EMs中疗效显著。初步的基础研究发现，该方能抑制子宫内膜MSCs向肌成纤维细胞分化，促使其向上皮细胞分化，作用机制可能和Wnt/β-catenin信号通路有关。本研究拟采用免疫印迹、qPCR、免疫荧光、细胞转染等实验技术，通过体内外研究，探讨Wnt/β-catenin通路与温肾消癥汤调控子宫内膜MSCs分化的关联程度，并揭示温肾消癥汤作用于Wnt/β-catenin信号通路的具体环节。研究结果将为温肾消癥汤临床治疗EMs提供科学依据，也为今后寻找新的EMs治疗方法提供理论基础。

子宫内膜异位症（EMs）作为妇科常见病、多发病，发病机制不明，临床缺乏有效的根治手段。异位病灶中存在的纤维化组织是引起EMs多种临床症状的重要原因。近年来越来越多的研究证据表明EMs是一种干细胞疾病。子宫内膜中的MSCs随经血逆流到达腹腔，是产生EMs的“种子”。MSCs在纤维化过程中发挥关键作用，多种组织来源的MSCs可分化为肌成纤维细胞，促进纤维化的发生发展。因此，阻断子宫内膜MSCs参与纤维化发展的途径可能为EMs的治疗提供新的方向。在本研究中我们分别分离、培养非子宫内膜异位症患者正常内膜MSCs和子宫内膜异位症患者在位内膜MSCs、异位内膜MSCs,并经细胞表面表型鉴定、多向分化潜能鉴定符合MSCs的特征。经比较，异位内膜MSCs表达纤维化标记蛋白collagen I、α-SMA、fibronectin和CTGF的水平高于正常、在位内膜MSCs，而且更易向肌成纤维细胞分化。子宫内膜MSCs分化为子宫内膜间质成纤维细胞后表达更高水平的collagen I、α-SMA、fibronectin和 CTGF。与之对应的是，异位内膜组织的纤维化程度显著高于在位、正常内膜，而且异位病灶的纤维化程度随着EMs的病程进展逐渐加重。进一步研究发现，异位子宫内膜MSCs中Wnt/β-catenin通路活化程度高于正常、在位内膜MSCs。子宫内膜MSCs中Wnt/β-catenin被激活时，纤维化标记蛋白表达增强；Wnt/β-catenin受抑制时，纤维化标记蛋白表达降低。温肾消癥汤干预后，异位子宫内膜MSCs中纤维化标记蛋白collagen I、α-SMA、fibronectin和CTGF表达水平显著下降，β-catenin总蛋白及活化状态的β-catenin表达水平显著降低，β-catenin的降解复合体重要成员GSK-3β的磷酸化水平显著下降，Axin表达明显上调，而且β-catenin的入核转运受到明显抑制。在EMs小鼠模型中进一步验证了温肾消癥汤对EMs病灶纤维化的治疗作用及对wnt/β-catenin信号通路的抑制作用。研究结果为阐明温肾消癥汤治疗EMs的作用机制提供重要的理论支持，也为EMs的治疗提供新的思路。