基于绵羊角质形成细胞探究SHH通过Krox20调节IGFBP5的表达影响羊毛弯曲

doi:10.11843/j.issn.0366-6964.2023.06.016

Abstract

Abstract: 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-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.

Key words: SHH, wool bending, sheep keratinocytes, Krox20, IGFBP5

CLC Number:

S826.2

DONG Yajie, HAO Xiaojing, WU Jinqiang, WANG Rong, ZHANG Pengxiang, WANG Haidong, HE Xiaoyan. Exploration of the Effect of SHH on Wool Bending through Krox20 Regulation of IGFBP5 Expression Based on Sheep Keratinocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2365-2375.

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Exploration of the Effect of SHH on Wool Bending through Krox20 Regulation of IGFBP5 Expression Based on Sheep Keratinocytes

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