摘要
本研究旨在探究音猬因子(Sonic hedgehog,SHH)与羊毛弯曲形成的关系及其调控机制。本研究通过培养原代绵羊角质形成细胞,构建过表达和沉默载体,利用细胞转染、免疫共沉淀、实时荧光定量PCR和Western blot等方法探究SHH与羊毛弯曲的关系,分析SHH是否通过早期生长应答基因2(early growth response 2,EGR2/Krox20)调控胰岛素样生长因子结合蛋白5(insulin-like growth factor binding proteins 5,IGFBP5)来影响羊毛弯曲。结果显示,体外成功分离培养绵羊原代角质形成细胞,显微镜下呈“铺路石”样,免疫荧光鉴定结果呈阳性表达,CCK-8结果表明细胞生长曲线呈“S”型,符合细胞增殖趋势;过表达和沉默SHH后,羊毛弯曲相关基因KRT71、β-catenin和TCHH的表达量显著升高和降低;过表达Krox20后,羊毛弯曲相关基因的表达量显著升高;免疫共沉淀试验表明,在绵羊角质形成细胞中SHH可以与Krox20相互作用且正调控Krox20;实时荧光定量PCR和Western blot结果显示,过表达SHH后显著上调IGFBP5的相对表达量,沉默SHH后显著下调IGFBP5的相对表达量;过表达Krox20后IGFBP5的相对表达量显著升高。本研究表明,在绵羊角质形成细胞中SHH通过Krox20调控IGFBP5来影响羊毛弯曲,结果为毛发弯曲性状形成的相关分子调控机制提供了理论依据。
The purpose of this study was to investigate the relationship between Sonic hedgehog(SHH)and wool bending formation and its regulatory mechanism.In this study,primary sheep keratinocytes were cultured,overexpression and silencing vectors were constructed,and cell transfection,immunocoprecipitation,quantitative real-time PCR and Western blot were used to explore the relationship between SHH and wool bending,and to analyze whether SHH regulates insulin-like growth factor binding proteins 5(IGFBP5)through early growth response 2(EGR2/Krox20)to affect wool bending.The results showed that sheep keratinocytes were successfully isolated and cultured in vitro,and were like"paving stone"under the microscope.Immunofluorescence identification results showed positive expression.CCK-8 results showed that the growth curve of sheep keratinocytes was"S"shape,which was in line with the trend of cell proliferation.After overexpression and silencing of SHH,the expressions of fleece fleece-related genes KRT71,β-catenin and TCHH were significantly increased and decreased.After overexpression of Krox20,the expression of wool bending related genes was significantly increased.Co-immunoprecipitation assay showed that SHH could interact with Krox20 and positively regulate Krox20 in sheep keratinocytes.The results of real-time PCR and Western blot showed that overexpression of SHH significantly up-regulated the relative expression of IGFBP5,while silencing of SHH significantly down-regulated the relative expression of IGFBP5.The relative expression of IGFBP5 increased significantly after overexpression of Krox20.The results indicate that SHH affects wool bending through Krox20 regulation of IGFBP5 in sheep keratinocytes,which provides a theoretical basis for the related molecular regulation mechanism of hair bending traits.
作者
董亚洁
郝晓静
吴晋强
王荣
张鹏翔
王海东
赫晓燕
DONG Yajie;HAO Xiaojing;WU Jinqiang;WANG Rong;ZHANG Pengxiang;WANG Haidong;HE Xiaoyan(College of Animal Medicine,Shanxi Agricultural University,Taigu 030801,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2023年第6期2365-2375,共11页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
山西省自然科学基金(201701D121106)。
