人脂肪源性间充质干细胞成软骨分化中miRNA-381与SOX9协同调控的机制研究

The early degeneration of tissue engineering cartilage, hope for the repairing of cartilage defect, after chondrogenesis of seed cell is a great trouble for its clinical application. The key point of homeostasis maintainance in chondrocyte is cell regulation. We have found that transcriptional and post-transcriptional regulation were both involved in the chondrocyte differentiation. Our new finding revealed that miR-381 was selectively expressed during early stage of chondrogenesis in human adipose-derived stem cells (hADSCs). Bioinformatics indicated that miR-381 was targeted to SOX9, a cartilage master transcription factor closely related to chondrocyte development. In this study, we focus on the mechanism of hADSCs chondrogenesis by ordered combinatorial regulation of miR-381 and SOX9 to improve chondrogenesis at transcriptional and post-transcriptional level. Firstly, we will observe the expression kinetics of miR-381, SOX9 and some specific biomarker of cartilage to figure out their regulative rules and relationships with chondrogenesis. Next, we will clarify circulating co-regulation rules of these factors during different stages of hADSCs chondrogensis. Finally, we will prove the rule of chondrogenesis by regulation of this co-regulation mechanism between miR-381 and SOX9, and provide fundamental basis for the control and improvement during chondrogenesis of seed cells in tissue engineering cartilage.

组织工程化软骨为软骨缺损修复带来曙光，但种子细胞成软骨诱导后较早肥大退变仍掣肘其向临床应用的转化，而维持软骨细胞内稳态的关键是细胞调控。我们已研究证实转录和转录后协同调控在维持软骨细胞内环境稳定中起重要作用，我们在hADSCs成软骨分化的miRNA表达谱基因芯片筛选及验证中发现了miR-381的特异性表达，生物信息学分析提示与软骨形成与分化密切相关的转录调控因子SOX9为其预测靶基因。本课题以miR-381和SOX9为切入点，研究转录与转录后调控子在hADSCs成软骨分化中协同调控的具体机制。通过观察hADSCs成软骨分化不同阶段miR-381、SOX9和软骨特征指标的表达动力学规律，研究miR-381和SOX9在hADSCs成软骨分化不同阶段的协同调控机制，阐明两者协同调控促进hADSCs定向成软骨分化的规律，为定向调控干细胞成软骨分化及优化组织工程化软骨的种子细胞奠定基础。

组织工程化软骨为软骨缺损修复带来曙光，但种子细胞成软骨诱导后较早肥大退变仍掣肘其向临床应用的转化，而维持软骨细胞内稳态的关键是细胞调控。我们已研究证 实转录和转录后协同调控在维持软骨细胞内环境稳定中起重要作用，我们在 hADSCs 成软骨分化的miRNA表达谱基因芯片筛选及验证中发现了miR-381的特异性表达，生物信息学分析 提示与软骨形成与分化密切相关的转录调控因子 SOX9 为其预测靶基因。因此，我们以 miR-381 和 SOX9 为切入点，研究转录与转录后调控子在 hADSCs 成软骨分化中协同调控的具体机制。通过体外实验以qRT-PCR检测IL-1β刺激后的人及鼠原代软骨细胞及ATDC5成软骨诱导后的软骨样细胞中的miR-381表达情况;体内实验以原位杂交技术检测鼠胚胎及骨关节炎患者软骨中的miR-381表达情况。构建RNA模拟物或抑制物评估miR-381在成软骨分化中的作用。荧光素酶报告基因检测miR-381在调节NF- kB信号通路中的作用，通过siRNA或过表达质粒定位上游的调节子Sox9及Runx2。结果提示miR-381在成软骨分化及肥大的ATDC5细胞中表达升高。IL-1β可诱导ATDC5、人及鼠原代软骨细胞中miR-381的表达上调。原位杂交后可见miR-381表达于鼠胚胎及骨关节炎患者的软骨退变吸收区域。过表达Runx2或Sox9后的ATDC5细胞中，miR-381表达增加。miR- 381可抑制II型胶原的表达及增加MMP13的表达，但并不调控NFKBIA 及NKRF。因此，miR- 381在成软骨分化及关节炎软骨中均高表达，它可能调节Sox9及Runx2，并通过抑制II型胶原和上调MMP13而在软骨基质吸收中起重要作用。本课题初步探讨了miR-381和SOX9在hADSCs成软骨分化不同阶段的协同调控机制，阐明两者协同调控促进 hADSCs 定向成软骨分化的规律，为定向调控干细胞成软骨分化及优化组织工程化软骨的种子细胞奠定基础。