肉鸡胫骨软骨发育不良生长板软骨细胞差异表达miRNAs筛选及其机制研究

Tibial chondrodysplasia (TD) is one of the severe bone disorders affecting broilers production. MiRNAs play an important role in the development of mammal skeletal diseases. In this study, microRNA chip technology will be used to isolate miRNAs from tibial growth plates of normal broilers, natural TD birds and thrium-induced TD broilers. The differential expressions of miRNAs will be verified by RT-qPCR and Northern blot analyses. Expression vectors of three differentially expressive miRNAs will be constructed and transfected into the chondrocytes from tibial growth plates in vitro. The effect of differentially expressed miRNAs on chondrocyte differentiation and development will be studied. The target genes of miRNA were predicted by the methods of bioinformatics, then screened and verified by miRNA pull-down and dual-luciferase reporter system, respectively to determine the role of miRNAs in the pathogenesis of TD. Based on the above outcomes, the pathogenesis of TD can be understood with new insights and will provide new ideas for prevention and treatment of TD or new anti-TD poultry breeding.

肉鸡胫骨软骨发育不良（TD）是一种影响肉鸡生产的骨骼疾病。miRNA在哺乳动物骨骼疾病的发生发展中具有重要作用。本研究利用微小RNA芯片技术筛选正常肉鸡、TD自然发病和福美双诱发TD肉鸡胫骨生长板特异性差异表达miRNAs，并采用RT-qPCR和Northen blot对差异表达的miRNAs进行验证，选取3条表达差异显著的miRNA构建表达载体，转染体外培养的肉鸡胫骨生长板软骨细胞，观察其对软骨细胞分化发育的影响；采用生物信息学方法预测潜在的靶基因，miRNA pull-down试验、荧光素酶报告系统筛选和验证靶基因，确定miRNAs在肉鸡TD发病中的作用，进一步明了肉鸡TD的发病机理，为TD的防治和抗肉鸡TD新品种的培育和提供新的思路。

miRNA在动物骨骼疾病的发展中具有重要的调控作用，然而其在肉鸡胫骨软骨发育不良的作用及调控机制尚不清楚。本项目围绕特异miRNA在肉鸡胫骨生长板软骨细胞增殖、分化和凋亡过程中的作用和机制开展了一系列的研究，取得以下进展：(1)完成了正常肉鸡与TD肉鸡生长板软骨细胞差异表达miRNA的筛选、分析了差异miRNAs的功能，利用生物信息学筛选出调控肉鸡TD发生的核心miRNAs(miR-140-5p,miR-460a)。(2)建立了肉鸡生长板原代软骨细胞分离培养的优化体系，明确了miR-140-5p与HDAC4的靶向关系。揭示了miR-140-5p靶向调控HDAC4，HDAC4抑制软骨细胞促分化转录因子(RUNX2,MEF2C)和软骨细胞肥大化蛋白(SOX9,Col-X,MMP-13,COMP)的表达，从而导致软骨细胞的生长与分化停滞。(3)明确了miR-460a与Bcl-2的靶向调控关系，揭示了miR-460a靶向抑制Bcl-2，并通过Caspase-3/Caspase-7途径介导TD肉鸡生长板软骨细胞的凋亡。本项目从miRNA水平解析了TD肉鸡发病时生长板软骨细胞肥大期分化停滞和软骨细胞异常凋亡的主要机理。阐明了关键miRNAs在肉鸡TD发病中的调控机制，同时本研究也表明miR-140-5p, miR-460a可作为肉鸡TD基因诊断和治疗的潜在靶点。