基于TGF-β/smads/ILK/GSK-3细胞信号通路探讨芪归益肾方延缓肾脏纤维化进展机制的实验研究

We have confirmed that the Chinese herbs of "decoction qigui yishen" can reduce urinary leakage of protein, decrease the aggregation of extracellular matrix (ECM) and lseeen the damage of podocyte. The prescription was set up by the principle of traditional Chinese medicine, which means that invigorating the spleen and kidney, promoting blood circulation to remove meridian obstruction. This research is in-depth study basing on the previous research. It is well known to all, fibrosis change has important pathology role to chronic kidney disease(CKD). Basing on the previous study of us and related literatures in recently years, we put forward the point of view that cell signal transduction pathways has the core action to the renal fibrosis. We want to use the idea 'regulation of cell signal transduction, delay the renal fibrosis progress' as the starting point to explore that why 'decoction qiguiyishen' can delays the renal fibrosis. Application with independent intellectual property rights of prescription, we will confirmed our ides and the efficiency of 'decoction qigui yishen' by the experiment In vivo and in vitro. The study of in vitro will base on the animal models, which has the similar lessions of CKD as the human disease. As to the experiments in vivo, we will use the mutil-level method to observe the cultivation of the renal tubular epithelial cells and the action of chinese herbs to it. We will observe that through regulating the cell signaling pathways of 'TGF-beta/smads /ILK/GSK-3' , the Chinese herbs maybe change the expression of cytokines, relieve the renal tubular interstitial transdifferentiation and nuclear factor, reduce the accumulation of ECM and affect the expression of the metabolic enzyme of ECM. In order to verify the efficacy and mechanism of traditional Chinese medicine is usefulto delay the course of renal fibrosis, we will use many advanced technology such as immune protein imprinting, fluorescent tracer technology and gene array,and so on.

已有工作证实以"健脾益肾，活血通络"为原则创立的中药方剂"芪归益肾方"对减少尿蛋白漏出、减轻细胞外基质聚集和保护足细胞方面的作用确切。本研究是在以往研究基础上的进一步深入。基于纤维化改变在慢性肾脏病病变进展中的重要作用，结合本课题组的研究基础和相关文献提出细胞信号转导通路在肾脏纤维化调控方面的核心地位。拟以"调控细胞信号转导，延缓肾脏纤维化进展"的视角，探讨"芪归益肾方"延缓肾脏纤维化的机制及有效性。应用具有自主知识产权的方剂"芪归益肾方"，以模拟人类病变情况的动物模型为基础，结合肾小管上皮细胞不同培养条件下的细胞学实验，观察调控"TGF-β/smads/ILK"细胞信号通路对细胞因子、细胞周期基因、肾小管间质转分化、核因子和细胞外基质及其代谢酶表达的作用。研究采用蛋白免疫印记、荧光示踪和基因芯片等技术手段验证中医药在延缓肾脏纤维化方面的机制及有效性。

本课题的研究工作按照国家自然科学基金申报书的工作安排主要以下几个方面的研究工作：1.进行TGF-β/smads/ILK/GSK-3信号转导通路与肾脏纤维化相关实验研究工作。2. 进行应用中药方剂及其组分在实验动物模型的药效学研究，筛选最佳的中药组分配伍及活性组分配比。3.进行中药方剂活性成分在延缓肾脏纤维化，保护慢性肾脏病模型动物肾脏功能的研究。4.利用中药制药工程学的方式开展中药复方及其活性成分新制剂的开发研究工作并取得了一定的进展。相关研究工作证实了芪归益肾方在慢性肾脏病中的治疗作用和保护机制。课题所用方剂获得国家发明专利授权保护(201510194881.X；ZL201610069692.0)。研究方剂在得到研究单位伦理委员会批准后开展临床研究工作验证疗效并进行成果转化工作。相关研究获得2015年度国家中医临床研究基地业务建设第二批科研专项（JDZX2015096）的资助。.研究工作开展四年以来，按照工作进度开展工作顺利，先后申报发明专利5项，授权发明专利2项；发表课题相关SCI论文7篇，北图核心期刊13篇，中文核心期刊1篇；培养硕士研究生3名；课题负责人被推选为中国中西医结合学会青年工作委员会副主任委员，中华中医药学会血液病专业委员会常务委员及内科专业委员会委员；课题工作先后获得中国中西医结合学会科技进步二等奖和江苏省中医药科技进步三等奖；课题相关的研究工作顺利完成。