龟板靶向维生素D受体促骨髓干细胞成骨分化作用整合机制

The key theory of traditional Chinese medicine (TCM) that kidney governed bones and generated marrow further has been studied. Based on our previous researcher achievement, we propose the hypothesis that effective components including steroids, fatty acids and their esters in Plastrum Testudinis ethyl acetate extracts (PTE) induced mesenchymal stem cell (MSCs) differetiation into osteoblast via vitamin D receptor (VDR) signaling pathway. The model in which PTE induced MSCs differetiation into osteoblast was developed based on the bone morphogenetic protein 4 (BMP4) promoter reporter gene construct, target gene of VDR signaling, we elucidate which response elements in target gene of VDR to effective components in PTE. The further studies used a wide variety of chromatin immunoprecipitation (ChIP)-based methods, co-immunoprecipitation and immunoblots, we elucidate which coregulators of VDR is involved in the effects of PTE-induced osteoblast differetiation. Using the RNAi-mediated knockdown VDR and mutagenesis of the functional domains in VDR, we demonstrate which functional domain of VDR is involved in the effects of PTE -induced osteoblast differetiation. In knockdown VDR mice, we elucidate the role of VDR signaling pathway in involving the effects of PTE -induced osteoblast differetiation. This study help not only to clarify the novel mechanism of VDR signaling pathway in involving the effects of Plastrum Testudinis-induced osteoblast differetiation, but also to solves the major problem which is to be study the differentiation of MSCs.

深入探讨中医"肾主骨生髓"理论, 基于前期研究成果，提出"龟板有效成分群以脂肪酸类、脂肪酸酯类和甾类结构特异性系列物形式存在，促骨髓间充质干细胞成骨分化是其相应系列物选择性作用于维生素Ｄ受体所产生的整合作用"假说。以龟板有效成分群促骨髓间充质干细胞（MSC）成骨分化为模型，针对维生素Ｄ受体(VDR)与其靶基因骨形态发生蛋白4（BMP4）。用基因启动子报告基因确定核受体靶基因对龟板有效成分群反应元件; 应用染色质共沉淀，免疫印迹和免疫共沉淀，阐明何种核受体辅激活子介导龟板有效成分群促成骨分化作用。以RNAi干扰VDR，或转染VDR与其受体功能区突变体到MSC中，阐明何种核受体功能区介导龟板有效成分群促成骨分化作用。应用VDR基因敲除鼠进一步确定VDR对龟板有效成分群成骨分化作用。为深入探讨中医益肾生骨治法提供新思路。

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。前期研究发现龟板提取物促进MSCs向成骨分化可能与其小分子脂肪酸类、脂肪酸酯类、甾体直接进入细胞膜作用内部核受体有关, 龟板提取物可上调MSCs定向成骨分化过程中VDR的表达。以龟板有效成分群促骨髓间充质干细胞（MSC）成骨分化为模型，构建了VDRE驱动的荧光素酶（Luc）报告基因系统，确定了龟板中十八酸乙酯，十四酸甾醇酯和4-甾酮为促MSC成骨分化的活性成分。以RNAi干扰VDR，或转染VDR与其受体功能区突变体到MSC中，发现4甾酮（S9）是作用于VDR的配体结合结构域（pCMV-Myc-VDR-C）而促进MSCs成骨分化的。miRNA基因芯片检测数据进行分析，我们发现经过龟板有效成分群、S9干预后，miRNA-330-3p、miRNA-200a-5p、miRNA-296-3p表达上调，同时miRNA-615、miRNA-351-3p、miRNA-129-1-3p、miRNA-466b-2-3p、miRNA-466b-1-3p表达下调。筛选出的miRNA-351模拟物可有效抑制VDR的表达，而在转染miRNA-351 抑制剂组则可促进VDR蛋白的表达，miRNA-351与VDR有相应的结合位点，能在其翻译水平有效抑制VDR的表达。通过lncRNA芯片技术分析龟板有效成分对lncRNA差异性表达情况，运用生物信息学方法，预测分析差异性表达的lncRNA共表达基因，预测分析其共表达的靶基因，运用生物学功能聚类分析的方法了解其参与成骨分化的相关情况，通过trans和cis预测分析差异性lncRNA参与基因的调控机制，构建TF-lncRNA-靶基因三者的网络关系。 证实“龟板有效成分群-miRNA-维生素Ｄ受体-lncRNA”的多元调控网络调控成骨分化。为中医临床益肾治法提供理论依据，优化以中医益肾理论为基础的创新中药提供思路。研究成果已发表 SCI 收录论文 9篇, 接受SCI 收录论文 1 篇,获得授权中药发明专利一项，完成1 名博士学位论文。