软骨脱细胞基质通过miR-140双向调节机制维持BMSC分化来源软骨内稳态的研究

Bone marrow mesenchymal stem cell（BMSC）has been widely used as seed cell for cartilage differentiation, however, although technology for induction in vitro is mature, build cartilages are tendency to ossification in vivo, it definitely hinders use of BMSC in clinic application,and which mechanism is not clear yet. Our previous studies have established a model for cartilage engineering using cartilage acellular matrix (CAM) and BMSCs, we found build cartilages could maintain their phenotype and histology in vivo. In accordance to with the studies that miR-140 plays an important part in cartilage differentiation and homeostasis, we infer CAM could maintain build cartilages homeostasis by stimulating up regulation of miR-140, on the one hand, increased miR-140 could excite expression of specific cartilage gene and protein, on the other hand , it simultaneously suppresses cartilage hypertrophy and ossification and balances secretion and degradation of cartilage matrix. The purpose of this study is to investigate the relationships between shape, histology, gene and protein expression of cartilage and bone and miR-140 in build cartilages constructed by CAM and BMSCs, and also we would demonstrate miR-140 mediated two-way regulation in remodeled matrix by microRNA suppression and overexpression. This study would give a promising way for solving the problems in clinic application of BMSC differentiated cartilage and supply the theoretical basis for cartilage construction by CAM.

骨髓间充质干细胞（BMSC）作为软骨分化的种子细胞被广泛应用，然而虽然体外诱导技术成熟，产物植入体内后仍易发生骨化，影响了其临床应用。前期实验我们发现应用软骨脱细胞基质（CAM）复合BMSC构建软骨体内能维持软骨结构及表型，结合以往的研究证实miR-140介导的转录后调控在软骨分化及维持稳定起关键作用，我们推测CAM可通过刺激BMSC上调miR-140来维持分化软骨体内的稳定。一方面，上调miR-140促进软骨特异性基质分泌；另一方面，抑制软骨肥大及软骨内骨化。本课题拟检测CAM对BMSC的miR-140表达影响及与构建软骨形态及组织学、软骨及骨相关基因及蛋白的相关性，同时利用抑制或过表达miR-140来阐明其在维持重建软骨稳定性的双向调节作用。本课题将从转录后水平解释CAM复合BMSC体内形成稳定软骨的机制，结果有望解决BMSC软骨分化的临床应用障碍，并为CAM应用于软骨构建提供理论依据

骨髓间充质干细胞（BMSC）作为软骨分化的种子细胞被广泛应用，但其植入体内后易发生骨化，构建物的弹性度降低影响了其临床应用。前期的实验我们发现应用软骨脱细胞基质（CAM）复合BMSC构建软骨体内能维持软骨结构及表型，结合以往的研究证实miR -140介导的转录后调控在软骨分化及维持稳定起关键作用，我们推测CAM可通过刺激BMSC 上调miR-140，从而维持分化软骨体内的稳定。一方面，上调miR-140促进软骨特异性基质分泌 ；另一方面，抑制软骨肥大及软骨内骨化。本课题的目的检测CAM对BMSC的miR-140表达影响及与构建软骨形态及组织学、软骨及骨相关基因及蛋白的相关性，同时利用抑制或过表达miR-140来阐明其在维持重建软骨稳定性的双向调节作用。本课题将从转录后水平解释CAM复合BMSC体内形成稳定软骨的机制。研究的主要内容(1) 软骨脱细胞基质复合BMSC构建软骨体外及体内形态及组织学、软骨和骨相关基因及蛋白的表达。(2) 软骨脱细胞基质复合 BMSC构建软骨体外及体内 miR-140表达水平的变化及与软骨、骨分化的相关性。(3)过表达或抑制 miR-140 对软骨分化及内稳态的影响对于已经在体内保持稳态的分化软骨。实验结果发现:(1)软骨脱细胞基质复合BMSC构建的组织在体外及体内能形成软骨并能表达软骨特异性蛋白Col II，并且植入体内后骨化程度较非CAM构建物低；(2)利用CAM复合BMSC构建软骨时miR-140表达升高；将BMSC过表达miR-140后，CCK-8检测其增殖速率无变化，但其构建组织软骨的相关基因ColII、SOX9表达增强，软骨肥大及骨相关Col I、Runx2、MMP13表达减弱；植入体内后过表达miR-140组能形成形态学和组织学较好的软骨组织。（3）多次利用sponge法抑制mir-140时发现并不能将BMSC的miR-140彻底抑制表达，其结果可能与BMSC的干细胞自身特性及内源性机制有关。本课题从转录后水平解释CAM复合BMSC体内形成稳定软骨的机制，为CAM 应用于软骨构建提供理论依据。