牦牛不同部位前体脂肪细胞分离鉴定及分化关键基因表达研究

doi:10.11843/j.issn.0366-6964.2022.03.009

摘要/Abstract

摘要： 旨在建立牦牛皮下和肌内前体脂肪细胞的体外研究模型,并检测两部位前体脂肪细胞分化过程中关键基因表达量差异,为研究牦牛不同部位脂肪沉积的分子机制提供试验材料和理论依据。本研究通过采取5头18~22月龄健康麦洼公牦牛的皮下脂肪组织和背最长肌组织,利用胶原酶消化,分离皮下和肌内前体脂肪细胞,随后根据细胞来源将细胞分为肌内组和皮下组,对其进行免疫荧光鉴定、生长曲线绘制、脂肪细胞油红O鉴定以及使用实时荧光定量技术检测分化关键基因PPARγ、C/EBPα、FASN、HSL的表达。结果表明,牦牛皮下和肌内前体脂肪细胞分离培养1 d时大部分为圆形,随培养时间增加,形态变为梭形;CCK-8结果显示生长曲线为正常的S型,并且皮下前体脂肪细胞在第4天生长速度显著高于肌内前体脂肪细胞;免疫荧光结果表明,PREF-1表达为阳性,表明分离的细胞确为前体脂肪细胞;分化后形成含有圆形大脂滴的成熟脂肪细胞,脂滴经油红O染色呈红色,且检测关键基因均有表达;PPARγ、FASN、HSL表达量在皮下和肌内前体脂肪细胞分化过程中的表达量随分化时间的增加而显著增加(P<0.05),肌内与皮下前体脂肪细胞中C/EBPα表达量在分化前期逐渐增加(P<0.05),在分化后期表达量出现下降,肌内前体脂肪细胞FASN和HSL表达量在第3天显著增加,而皮下前体脂肪细胞在第6天才显著增加。综上所述,本试验成功分离了牦牛皮下和肌内前体脂肪细胞,并成功诱导分化至成熟脂肪细胞,发现成脂分化关键基因PPARγ、HSL、FASN表达量在9 d分化期内均呈现上升趋势,C/EBPα表达量在分化第6天之后显著下降,为进一步研究牦牛皮下和肌内脂肪沉积的分子规律提供了参考。

关键词: 牦牛, 前体脂肪细胞, 不同部位, 分化关键基因

Abstract: The aim of this study was to establish an in vitro study model of subcutaneous and intramuscular preadipocytes in yak, and to detect the expression differences of key genes in the differentiation process of preadipocytes in the two parts, so as to provide experimental materials and theoretical basis for the study of the molecular mechanism of fat deposition in different parts of yaks. The subcutaneous adipose tissue and longissimus dorsi muscle tissue of 5 healthy male Maiawa yaks aged 18 to 22 months were collected, and the subcutaneous and intramuscular preadipocytes were separated by collagenase digestion. The cells were then divided into intramuscular group and subcutaneous group according to the cell sources. Immunofluorescence identification, growth curve mapping, Oil red O identification of adipocytes and real-time fluorescence quantitative technology were used to detect the expression of key differentiation genes PPARγ, C/EBPα, FASN and HSL. The results showed that yak subcutaneous and intramuscular adipocytes on the first day of cultivation were mostly circular, with the increase of incubation time, formed a spindle, the normal "S" growth curve model for CCK-8 detection, and growth rate of subcutaneous preadipocytes on day 4 was significantly higher than that of intramuscular preadipocytes. After identification by immunofluorescence, the expression of PREF-1 was positive, and they were identified as preadipocytes. After differentiation, mature adipocytes with large and round fat droplets were formed, and red fat droplets were detected by Oil red O staining, and the key genes were expressed. The expression levels of PPARγ, FASN and HSL during subcutaneous and intramuscular preadipocyte differentiation were significantly increased with the increase of differentiation time (P<0.05), the expression of C/EBPα in intramuscular and subcutaneous preadipocytes increased gradually at the prophase of differentiation (P<0.05), and the expression level was decreased at the later stage of differentiation. The expression of FASN and HSL in intramuscular preadipocytes showed significant increase on day 3, while in subcutaneous preadipocytes showed significant increase on day 6. In conclusion, subcutaneous and intramuscular preadipocytes of yak were successfully isolated and differentiated into mature adipocytes. Preliminary detection showed that the expression levels of key adipogenic differentiation genes PPARγ, HSL and FASN all showed an increasing trend during the differentiation period for 9 days, while the expression levels of C/EBPα decreased significantly after 6 days of differentiation. This study provided a reference for further studying the molecular rules of fat deposition in subcutaneous and intramuscular adipocytes in yaks.

Key words: yak, preadipocytes, different parts, differentiation of key genes

中图分类号:

S823.85

王森, 师俊华, 王之盛, 胡瑞, 王俊梅, 薛白, 彭全辉. 牦牛不同部位前体脂肪细胞分离鉴定及分化关键基因表达研究[J]. 畜牧兽医学报, 2022, 53(3): 755-765.

WANG Sen, SHI Junhua, WANG Zhisheng, HU Rui, WANG Junmei, XUE Bai, PENG Quanhun. Isolation and Identification of Preadipocytes from Different Parts of Yak and Expression of Key Genes for Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 755-765.

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