新型白术内酯促骨髓间充质干细胞向软骨分化的网络共激活机制

The key problem in the repair treatment of cartilage tissue defects is how to directly induce chondrogenic differentiation of mesenchymal stem cells (MSCs). In our previous work, we isolated a new atractylodes lactone (L) from Atractylodes macrocephalae used to invigorating spleen in traditional Chinese Medicine (TCM), under the guidance of the Strengthening Spleen theory in TCM. The new lactone L was found to promote MSCs chondrogenic differentiation via activating the transcription factor and microRNA in the differentiation progress. On this basis, we hypothesized that, “a new atractylodes lactone could induce the chondrogenic differentiation of MSCs through the integrated network mechanism of miRNA-transcription factor regulation”. In the project, the miRNA promoter report gene systems will be constructed to observe the effect of new atractylodes lactone on miRNA promoter activity in the cellular model of chondrogenic differentiation of MSCs. And miRNA-transfection method will be applied to the chondrogenic differentiation models in cells and animal to illuminate the concerted activation mechanism of new lactone L, which targets to feed-forward loop for directly chondrogenic differentiation of MSCs. The transcription factor promoter report gene system will also be constructed to investigate the effect of new lactone L on promoter activity. And RNAi or transcription factor-transfection method will be utilized in chondrogenic differentiation models in cells and animal to illustrate the feedback loop mechanism. Our study may provide a new idea for innovation of Chinese medicine herbs on the basis of Strengthening Spleen Theory in TCM, and a theoretical explanation for clinical treatment of strengthening spleen. Also it may play a foundational role in drug development and finding new target in bone diseases.

如何促骨髓间充质干细胞(MSCs)定向软骨分化，是修复、治疗软骨组织缺损的关键。在中医健脾理论指导下，我们从健脾中药白术中得到新型白术内酯L，它能激活软骨分化的转录因子和miRNA，促MSCs向软骨细胞分化。鉴此，我们提出“新型白术内酯以miRNA-转录因子调控网络的共激活机制,促MSCs向软骨分化"假说。本课题拟以MSCs软骨分化为模型，构建miRNA启动子报告基因系统，研究该内酯对启动子活性的影响；再转染miRNA到软骨分化细胞和动物模型中，阐明该内酯靶向前馈回路促MSCs向软骨分化的共激活机制；并构建转录因子启动子报告基因系统，探讨对转录因子启动子活性影响；用RNAi技术封闭转录因子(或转染转录因子到软骨分化细胞和动物中)，探求该内酯靶向反馈回路促MSCs向软骨分化的共激活机制。为中医健脾临床治法提供理论依据，为基于健脾理论的中药创新提供新思路，为开发骨病新药和发现新靶标奠定基础。

骨髓间充质干细胞(Bone mesenchymal stem cells, BMSCs)是目前研究最多、应用潜力最大的干细胞之一，是研究增殖与分化机理的理想模型。但是，骨髓中MSCs含量极少，长期增殖活性弱，扩增速度慢，逐渐向脂肪老化，无法满足大量的临床需求。因此，如何提高MSCs的成骨潜能是一个关键问题。在中医健脾理论指导下，我们从健脾中药白术中得到新型白术内酯L，它能激活软骨分化的转录因子和miRNA，促MSCs向软骨细胞分化。以新型白术内酯L干预骨髓间充质干细胞（BMSCs），通过miRNA-seq筛查成骨分化方面有关的miRNA 信息，发现miRNA-296在成软骨分化的过程中显著上调。转染miRNA-296模拟物及抑制物，诱导BMSCs成软骨分化，发现miRNA-296模拟物组可抑制成软骨分化相关标志基因的表达，而抑制物组可促进软骨分化相关标志基因的表达，证实miRNA-296可抑制成软骨的分化。以新型白术内酯L促骨髓间充质干细胞（MSC）成骨分化为模型，构建了miRNA-296启动子活性的荧光素酶（Luc）报告基因系统，确定了新型白术内酯L可靶向抑制miRNA-296启动子的活性。网站预测miRNA-296的靶基因，证实miRNA-296可靶向抑制Cyp26b1的表达。进一步构建Cyp26b1 3’UTR区荧光素酶报告基因系统，发现miRNA-296可靶向Cyp26b1 3’UTR区发挥作用。证实白术内酯L作用于miRNA-转录因子调控网络的共激活机制, 促MSCs定向软骨分化。为中医临床健脾治法提供理论依据，优化以中医健脾理论为基础的创新中药提供思路。研究成果已发表SCI论文 16篇，其中，收录论文 14篇，完成5名硕士学位论文。