ADAMTS18在软骨细胞分化中的作用及其机制研究

Chondrocyte differentiation plays a critical role in endochondral ossification and affects the growth and development of bone. Recent study suggests that some members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin-like motifs) metalloproteinase family play an important part in cartilage development and bone diseases. However, the exact roles of ADAMTS18 in chondrocyte differentiation and bone development still need to be further elucidated. The preliminary results demonstrate that ADAMTS18-deficient mice are small 2 weeks after birth and show early hypertrophic differentiation of growth plate, suggesting ADAMTS18 affects endochondral ossification through regulation of hypertrophic chondrocyte differentiation. Based on reports in the literature, we hypothesize that proteolytic activity of ADAMTS18 also plays an important role in chondrocyte differentiation. To do so, C3H10T1/2 cells and ADAMTS18-deficient mice are used to evaluate the exact role of ADAMTS18 in chondrocyte differentiation, and several methods including gene overexpression, gene knockdown and knockout are performed to study the role and molecular mechanism of ADAMTS18 in chondrocyte hypertrophic differentiation. Furthermore, yeast-hybrid system and site-specific mutagenesis are performed to study the correlation between its proteolytic activity and the effect of chondrocyte differentiation. In conclusion, our study will provide a new method and target for treatment of cartilage dysplasia and other related diseases, and also have important scientific significance and potential clinical prospect.

软骨细胞分化是软骨内成骨的核心，影响整个骨骼的生长发育。已有研究证实ADAMTS蛋白酶家族部分成员在软骨细胞分化中具有重要作用，而ADAMTS18是否影响软骨细胞分化尚不清楚。我们的前期研究发现ADAMTS18基因敲除小鼠在出生后2周出现体型明显偏小，生长板软骨细胞肥大分化提前，提示ADAMTS18可能通过调控软骨细胞肥大分化而影响软骨内成骨。结合文献报道，我们推测ADAMTS18蛋白酶活性在软骨细胞分化中也具有重要作用。为验证这些假设，本项目将以软骨细胞分化的细胞模型和ADAMTS18基因敲除小鼠为研究对象，利用过表达、基因沉默和敲除等方法探讨ADAMTS18在软骨细胞分化中的作用；利用酵母双杂交和定点突变技术研究ADAMTS18蛋白酶切活性对软骨细胞分化的影响。本项目将有助于加深对软骨发育异常等相关疾病的重新认识，为临床治疗提供新的思路和靶点，具有重要的科学意义。

自本项目立项以来，课题负责人及课题组成员按计划任务书的要求，围绕 “ADAMTS18在软骨细胞分化中的作用及机制研究”这一主题开展研究。主要研究内容包括通过体内、体外实验研究ADAMTS18在软骨细胞分化中的作用，利用酵母双杂交系统筛选ADAMTS18的底物，从而进一步研究ADAMTS18蛋白酶活性对软骨细胞分化的影响等。通过对上述内容的研究，主要取得了以下具有创新意义的研究结果：确定了ADAMTS18参与软骨细胞分化；构建了ADAMTS18过表达和敲除的稳定细胞株，证实了ADAMTS18抑制软骨细胞分化为肥大软骨细胞；利用酵母双杂交系统和免疫共沉淀确定了ADAMTS18的降解底物；利用定点突变技术分析ADAMTS18蛋白酶切活性对软骨细胞分化的影响；利用ADAMTS18基因敲除小鼠阐明了ADAMTS18 基因在软骨发育中的生物学功能。本项目将对软骨发育异常等相关疾病的研究提供重要的理论基础，为临床治疗相关疾病提供新的思路和靶点，具有重要的科学意义。