miR-17通过Fas-FasL通路调控牙周膜干细胞免疫调节特性的机制研究

Chronic inflammatory diseases, such as periodontitis and rheumatoid arthritis (RA), are the most common causes of bone tissue destruction involved in the immune response. Recently, stem cells become the hot spot in chronic inflammatory diseases treatment. Periodontal ligament tissue-derived mesenchymal stem cells (PDLSCs) have been isolated as multipotent and immunomodulatory potential stem cells that are capable of being used in stem-cell-mediated periodontal tissue regeneration.In our previous study, we have demonstrated that the miR-17 and Smurf1 were involved in a coherent feed-forward loop to regulate PDLSCs osteogenic differentiation. However, the exact effect and mechanism of miRNAs in PDLSCs immunomodulatory potential is not fully clarified. By microarray expression profiling of microRNA, we have found that miR-17 is necessary for PDLSCs immunomodulatory. Computational miRNA target prediction analyses were conducted, and we noted that FasL is one of the predicted targets of miR-17. On basis of our previous studies, in this study, firstly, we will further investigate the effect of miR-17 in immunomodulatory properties of PDLSCs. Secondly, we will explore the effect and mechanism of miR-17 on immunomodulation of PDLSCs via Fas-FasL pathway. Thirdly, we will observe the PDLSCs-mediated therapeutic effects in experimental periodontitis animal model. The elucidation of the molecular mechanisms will provide us with a better knowledge of miR-17 regulation in PDLSCs and improve stem-cell-mediated inflammatory bone disease therapy.

慢性炎症疾病如牙周炎和风湿性关节炎是与免疫相关的骨破坏疾病，利用干细胞控制炎症并促进骨再生是治疗此类疾病的研究热点之一。牙周膜干细胞（PDLSCs）具有多向分化和免疫调节的双重特性，是牙周组织修复再生的主要细胞群。我们前期研究发现miR-17通过Smurf1形成反馈环路负向调控PDLSCs成骨分化。但miRNAs在PDLSCs免疫调节中的具体作用及分子机制尚不明确。通过芯片筛选我们发现miR-17仍是PDLSCs免疫调节中关键miRNA；生物信息学分析发现FasL是miR-17重要的靶基因。本项目拟在前期研究的基础上，通过报告基因检测、细胞共培养、基因沉默、RNA干扰、micro-CT等研究方法从体内和体外深入探讨miR-17活化Fas-FasL通路增强PDLSCs免疫调节特性的机理，为阐明miR-17在PDLSCs中的调控功能及为控制炎症促进骨再生的免疫相关骨破坏疾病的治疗提供依据。

牙周炎是一种和免疫相关的慢性骨破坏疾病，控制炎症并促进骨形成是此类疾病治疗的核心问题。牙周膜干细胞（PDLSCs）具有多向分化和免疫调节的双重特性，是牙周组织修复再生的主要细胞群。前期研究我们发现miRNA能调控PDLSCs骨向分化。但miRNAs在PDLSCs免疫调节中的具体作用及分子机制尚不明确。因此本项目中我们通过细胞共培养、芯片筛选、报告基因检测、基因过表达、RNA干扰、micro-CT等研究方法从体外和体内深入探讨miR-17在PDLSCs免疫调节特性中的作用和机理。. 为了筛选确定PDLSCs免疫调节特异性miRNA及靶基因，我们将PDLSCs与PBMCs共培养，体外建立免疫炎症微环境，再通过miRNA芯片检测发现miR-17是PDLSCs免疫调节中关键miRNA；生物信息学预测和实验均验证FasL是miR-17调控PDLSCs免疫调节特性的重要靶基因。为明确miR-17和FasL在PDLSCs免疫调节特性中的作用和机制，我们通过免疫荧光、Realtime PCR、和Western Blot证实PDLSCs表达FasL；利用细胞转染miR-17，发现过表达miR-17，靶基因FasL表达降低，共培养液及PDLSCs中可溶性免疫调节因子IDO 和TGF-β表达减少，相反PBMCs的增殖活性增加，共培养液中炎性因子含量增加。沉默miR-17，靶基因FasL表达增加，共培养液及PDLSCs中可溶性免疫调节因子IDO 和TGF-β表达增加，而共培养的PBMCs增殖活性降低，共培养液中炎性因子含量减少。转染miR-17的PDLSCs移植入大鼠牙周炎动物模型，与体外细胞实验结果相一致。以上研究结果表明miR-17能够通过Fas-FasL通路减少PDLSCs可溶性免疫调节因子的分泌，并影响PBMCs的活性，从而实现免疫调控。