gga-miR-1600靶向MnSOD调控鸡缺锰性胫骨软骨发育不良的分子机制

锰缺乏可致鸡胫骨软骨发育不良，氧化应激与自噬在软骨细胞增殖分化中发挥重要作用。前期研究发现，缺锰鸡胫骨软骨中锰超氧化物歧化酶(MnSOD)基因表达显著下调和gga-miR-1600表达显著上调，但gga-miR-1600与MnSOD的关系及二者在胫骨生长板软骨细胞增殖分化中的作用尚不明确。本项目在复制鸡锰缺乏症模型和建立gga-miR-1600敲低与过表达的鸡胫骨生长板软骨细胞模型的基础上，应用双荧光素酶报告基因技术明确gga-miR-1600与MnSOD的靶向关系；应用荧光定量PCR法、免疫印迹技术、激光共聚焦及流式细胞术检测II型胶原、X型胶原、转录因子Nrf2通路中关键信号分子的表达，氧化应激及自噬相关指标，旨在阐明gga-miR-1600靶向MnSOD介导氧化应激与自噬在鸡缺锰性胫骨生长板软骨细胞增殖分化障碍中的作用机制，为研究鸡锰缺乏症提供新的分子靶标及其理论依据。

Manganese deficiency can cause tibial chondrodysplasia in chickens, and oxidative stress as well as autophagy play important roles in the process of proliferation and differentiation in chondrocytes. In the previous studies, we found that the expression levels of manganese superoxide dismutase (MnSOD) gene and gga-miR-1600 gene was significantly down-regulated and up-regulated by manganese deficiency in.chickens, respectively. However, the relationship between gga-miR-1600 and MnSOD with their roles in the proliferation and differentiation of chondrocytes in the tibial growth plate remain to be clarified. In this study, we will establish the model of manganese deficiency in chickens, i.e., in vivo studies, and chondrocytes of chicken tibial growth plate with knockdown and overexpression of gga-miR-1600, i.e., in vitro studies. Double luciferase reporter gene detection will be performed to clarify the targeting relationship between gga-miR-1600 and MnSOD in vitro models. Simultaneously, qPCR method, western blot analysis, confocal fluorescent microscopy and flow cytometry will be applied to assess the expression levels of type II collagen, type X collagen and key signaling molecules in the Nrf2 pathway, as well as assays related to oxidative stress and autophagy in vivo and in vitro models. This study will clarify the molecular mechanism of gga-miR-1600 targeting MnSOD-mediated oxidative stress and autophagy in the disorder of proliferation and differentiation in tibial growth plate induced by.manganese deficiency in chickens, which will provide a new molecular target and important theoretical basis for the study of chicken manganese deficiency.