miR-17-92的差异性表达在骨愈合中的作用和机制研究

The process of bone healing is similar to bone development, and it is regulated by chondrocyte, osteoblast and osteoclast. Our preliminary studies show that miR-17-92 cluster could regulate bone development and remodeling, and the expression level of miR-17-92 cluster in bone nonunion group is lower than normal group. These results suggest miR-17-92 cluster may regulate bone healing. Our results show that homozygous deletion of miR-17-92 cluster in chondrocyte, osteoblast and osteoclast result in quiet different bone phenotypes. The expression level of miR17-92 cluster in different phase of bone healing is different too. These data suggest that differentially expressed miR-17-92 cluster is an important model for miR-17-92 cluster to regulate bone healing. In this study, animal model of bone nonunion will be established to explore the relationship between miR-17-92 cluster and bone healing. The fracture model of differentially expressed miR-17-92 cluster will be established to clear the key phase and cell which are regulated by miR-17-92 cluster during bone healing. The cell model of differentially expressed miR-17-92 cluster will be established to explore the molecular mechanism in regulating chondrocyte, osteoblast and osteoclast function. This research will reveal the impact and mechanism of differentially expressed miR-17-92 cluster in bone healing, and supply a new strategy for early identification and treatment of bone nonunion.

骨愈合与骨发育过程相似，软骨、成骨和破骨细胞主导其进程。我们前期研究证实miR17-92调控骨发育和重塑；骨不愈合患者血液中miR17-92的表达较正常愈合患者显著降低；提示miR17-92可能影响骨愈合。课题组发现软骨、成骨及破骨细胞内特异敲除miR17-92小鼠骨表型存在明显差异；C57小鼠骨折后不同时段miR17-92的表达也不相同；综上提示miR17-92在骨折后不同细胞和时段的差异表达可能是其调控骨愈合的重要方式。课题组拟通过骨不愈合临床病例和动物模型，阐明miR17-92与骨愈合的相关性；构建骨折后不同细胞和时段内差异表达miR17-92的动物模型，明确其调控骨愈合的关键靶细胞和时段；构建miR17-92差异表达的细胞模型，探索其调控软骨、成骨和破骨细胞功能的分子机制。本课题旨在揭示miR17-92的差异表达在骨愈合中的作用和机制，为骨不连的早期诊断和促进骨愈合的治疗提供新思路。

探索骨愈合的调控机制是骨科研究热点之一。课题组设想miR-17-92可能通过调控各骨功能细胞（软骨、成骨、破骨和骨细胞）影响骨发育、重塑及骨愈合。据此，课题组研究如下：1、miR-17-92与骨愈合进程和质量的相关性研究；2、骨功能细胞对miR-17-92对骨形成/愈合的影响；3、miR-17-92对骨功能细胞的调控作用。结果如下：1、miR-17-92与骨愈合有关。2、与野生型小鼠（WT）比较，软骨细胞内敲除miR-17-92小鼠（miR-17-92-ccKO）体型无差异，骨量升高，骨强度升高，干骺端内Sox9、Col2a、Col10a、Runx2、ALP、Col1a、CTSK、OPG及RankL表达升高，股骨骨小梁表面破骨细胞增多，miR-17-92-ccKO小鼠骨折后15、21天时骨痂面积更大；骨折后10、15、21天时骨愈合评分更高，miR-17-92-ccKO小鼠骨愈合加快。骨折后15天，miR-17-92-ccKO小鼠骨折断端骨痂内硬骨痂明显增多；骨折后21天，断端周围软骨痂由编织骨取代，进入骨痂重塑期。成骨细胞内敲除miR-17-92小鼠（miR-17-92-OBKO）骨量下降，成骨功能下降。破骨细胞内敲除miR-17-92小鼠（miR-17-92-OCKO），骨量增大，破骨功能降低。骨细胞内敲除miR-17-92小鼠（miR-17-92ocyKO），无骨骼畸形，骨量下降，骨强度下降，破骨功能下降。3、miR-17-92调控小鼠软骨及ATDC5软骨祖细胞功能存在差异和时间窗，促进软骨发育，通过TGFβ通路增强软骨细胞功能。miR-17-92负调节成骨分化，成骨细胞内miR-17-92表达下降抑制成骨功能。miR-17-92负调节破骨分化和成熟，CTHRC1可能与miR-17-92-OCKO小鼠成骨增强相关，miR-17-92可能作用于CTHRC1调节成骨功能。miR-17-92可能靶向Tgfbr2基因，调控骨细胞内TGFβ信号通路，促进骨细胞增殖，减少RANKL表达，抑制破骨功能。此外，由于髓核细胞是类软骨细胞，我们观测了miR-17-92在椎间盘退变中的调控作用，发现miR-17-92靶向Kif26b通过Kif26b/PI3K/Akt通路促进髓核细胞凋亡。上述结果丰富了骨形成、骨愈合及椎间盘退变的分子机制，为相关疾病的诊疗提供了新的思路。