山羊毛乳头细胞分离鉴定及TGF-β/smad通路相关基因的表达

doi:10.16431/j.cnki.1671-7236.2020.09.004

摘要/Abstract

摘要： 试验旨在分离山羊毛乳头细胞，并探索其中TGF-β/smad通路相关基因的表达，为体外开展山羊毛囊发育的相关机制研究提供良好模型。采用中性蛋白酶与胶原酶两步消化法对山羊背部皮肤做处理，在体视显微镜下分离毛乳头细胞并进行纯化。细胞免疫荧光和Western blotting验证平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）和波形蛋白（vimentin，VIM）在体外培养的毛乳头细胞中的表达，并检测TGF-β/smad通路中相关基因在山羊毛乳头细胞中的表达情况。结果显示，分离后进行贴壁培养的毛乳头细胞生长较慢，在分离15 d后具有成熟形态，可进行传代培养。细胞免疫荧光和Western blotting结果表明，α-SMA和VIM在体外培养的毛乳头细胞中均表达，TGF-β/smad通路中smad4、smad5基因的表达水平显著高于对照组（P<0.05），smad2、smad6、smad7基因的表达量极显著高于对照组（P<0.01），这些与毛囊发育相关的关键基因均呈现高表达，表明毛乳头细胞可能通过TGF-β/smad通路的调控从而影响毛囊的发育。本研究成功分离出山羊毛乳头细胞，为体外研究山羊毛囊发育机制奠定良好的理论基础和细胞模型。

关键词: 山羊; 毛乳头细胞; 酶消化法; TGF-β/smad; 毛囊发育

Abstract: The purpose of this study was to isolate goat hair follicle cells,and to explore the expression of TGF-β/smad pathway-related genes,providing a good model for the research on the mechanism of goat hair follicle development in vitro.In this study,two steps of neutral protease and collagenase digestion were used to treat the skin on the back of goats,the hair follicle cells were isolated and purified under stereomicroscope.Cellular immunofluorescence and Western blotting method were used to verify the expression of α-SMA (α-smooth muscle actin,α-SMA) and VIM (vimentin,VIM),and the expression of related genes of the TGF-β/smad pathway in goat hair papilla cells were examined.The results showed that the isolated goat hair follicle cells in adherent culture after separation grew slowly and had mature morphology after 15 days of separation and could be subcultured.The results of immunofluorescence and Western blotting showed that both α-SMA and VIM were positive in vitro cultured hair papilla cells.The expression of smad4 and smad5 genes in the TGF-β/smad pathway were significantly higher than those in control group (P<0.05),and the expression of smad2,smad6,and smad7 were extremely significantly higher than those in control group (P<0.01).These key genes related to hair follicle development were highly expressed.This study successfully isolated goat dermal papilla cells,which would give a good theoretical basis and cell model for studying the hair follicle development mechanism in vitro.

Key words: goat; hair papilla cells; enzymatic digestion; TGF-β/smad; hair follicle development

中图分类号:

S827

丁洋洋, 赵会静, 关伟军, 何晓红, 浦亚斌, 蒋琳, 马月辉, 赵倩君. 山羊毛乳头细胞分离鉴定及TGF-β/smad通路相关基因的表达[J]. 中国畜牧兽医, 2020, 47(9): 2724-2731.

DING Yangyang, ZHAO Huijing, GUAN Weijun, HE Xiaohong, PU Yabin, JIANG Lin, MA Yuehui, ZHAO Qianjun. Isolation and Identification of Goat Dermal Papilla Cells and Expression of TGF/β-smad Pathway Related Genes[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(9): 2724-2731.

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