miR-214通过调控间充质干细胞自我更新影响免疫微环境中骨再生及其相关机制研究

Mesenchymal stem cell (MSC) has been an important participant in the process of bone regeneration due to its multi-differentiation capacity. In addition, MSC supplies abundant seed cells for bone regeneration by consistent self-replication, namely “self-renewal”. MicroRNA-214 is one of recently identified bone-related microRNAs. In spite of its well understanding in the maturation of osteocytes, the modulatory effect of miRNA-214 on MSCs remains unknown. Based on our previous findings and the sequencing technology, we discovered that miR-214 modulates the self-renewal of MSC in immune microenvironments. Furthermore, the miR-214 deficient mouse exhibits osteodysplasty. In addition, the colony forming capacity of the mouse bone derived MSC was impaired and the effect was closely bound up with the expression of some transcription factors including Nanog，OCT-4 and SOX-2. However, the study about the regulatory effects on MSC self-renewal and the related mechanisms have not been reported. Hence, this proposed study will mechanistically investigate the influence of miRNA-214 on bone regeneration in immune microenvironments by regulating the self-renewal of MSC through studying the model of immune-related bone destruction and bone fracture. This study will shed light on the comprehensive understanding of modulatory effect of miR-214 on MSC and the bone regeneration in immune microenvironments; thus contribute to theoretical accumulation in the field of bone regeneration and provide new theoretical guidance for the clinical application of MSC.

间充质干细胞（MSC）是骨再生的重要参与者，它既能分化为多种子代细胞，参与修复骨组织，又能不断自我更新，为骨再生提供充足的种子。miR-214是新发现的骨相关微小RNA之一，以往研究侧重其对成骨细胞的调节，有关其对MSC的作用报道不多。我们在前期工作基础上，通过测序技术筛选意外发现：miR-214参与调节免疫微环境中MSC的自我更新能力。敲除miR-214后，小鼠骨发育异常，骨来源的MSC集落形成能力受损，且与转录因子Nanog, OCT-4和Sox-2的表达密切相关。而目前国内外研究未见miR-214调节MSC自我更新能力及其机制的报道。为此，本课题拟采用基因敲除鼠建立免疫骨损伤和骨折模型，深入研究miR-214对MSC自我更新能力的调节和对免疫微环境中骨再生的影响及机制。本课题有助于全面了解miR-214对MSC的调控作用，将为骨再生研究提供理论积累，为MSC临床应用提供新的理论依据。

干细胞自我更新是指干细胞自我复制的能力，维持干细胞适当的自我更新可以维持干细胞数量的稳定，为组织再生提供源源不断的种子。MiR-214是调节骨发育分化的重要小RNA之一，但是其对于MSC的自我更新活动的调控作用却报道不多。本课题分析了miR-214敲除小鼠的MSC 自我更新能力；其次建立起了稳定高表达miR-214的MSC细胞株以及cas-9技术介导的miR-214稳定敲除MSC 细胞株；再次，采用高通量技术分析了miR-214过表达和敲除以后MSC内关键基因（col-II, pten等）表达的变化。此外，我们还评价了miR-214过表达和敲除以后MSC体内外的骨-软骨再生效果。结果表明：miR-214有助于维持MSC 的自我更新能力，但是miR-214敲除有利于MSC骨软骨再生修复作用。我们还发现miR-214还可以通过调节MSC分泌免疫因子影响其免疫调节功能等重要功能。