哈萨克羊胎儿期臀脂前体脂肪细胞的增殖和分化特性研究

doi:10.16431/j.cnki.1671-7236.2025.10.004

摘要/Abstract

摘要： 【目的】分离哈萨克羊臀脂形成关键窗口期的前体脂肪细胞，从组织学和细胞学角度掌握哈萨克羊臀脂沉积的发育性特征，为深入研究绵羊臀脂沉积的分子机制奠定基础。【方法】采集哈萨克羊妊娠第50(E50)、60(E60)、70(E70)、80(E80)、94(E94)和105(E105)天的胎儿，观察其臀脂形态变化，并对E70、E80及E94时期胎儿的臀脂组织进行油红O染色及成脂分化相关基因的表达检测，确定哈萨克羊臀脂沉积的关键时期。利用Ⅰ型胶原酶分段消化法分离E70、E80和E94时期哈萨克羊臀脂前体脂肪细胞，连续培养6 d以绘制细胞生长曲线，比较3个胎儿期来源的前体脂肪细胞增殖能力。对E70、E80和E94时期哈萨克羊臀脂前体脂肪细胞进行成脂诱导分化，通过油红O染色、脂滴含量测定及成脂分化相关基因表达水平的实时荧光定量PCR检测，比较不同胎儿期获得的前体脂肪细胞的成脂分化特性。【结果】哈萨克羊E70时期出现明显的脂臀形态，E80时期臀部组织开始出现明显的脂滴，并在E94时期脂滴积累增多。成脂分化相关基因表达量随妊娠时间逐渐增加，且在E80～E94时期呈倍数增加。通过Ⅰ型胶原酶分段消化法分离获得E70、E80和E94时期的哈萨克羊臀脂前体脂肪细胞，细胞形态均呈成纤维细胞样，生长活力良好；3个时期的前体脂肪细胞经诱导分化处理后均有成熟脂滴生成，且成脂分化相关基因CEBPα、PPARγ、LPL和FABP4表达量较诱导分化前均显著增加(P＜0.05)。E80时期前体脂肪细胞在诱导分化第3天即形成脂滴，相较于E70和E94时期脂滴形成时间最短、含量最高。【结论】本研究分离获得的哈萨克羊E70、E80和E94时期胎儿臀脂前体脂肪细胞增殖活力良好，可诱导分化形成成熟脂滴，且E80时期臀脂前体脂肪细胞分化能力更强。

关键词: 哈萨克羊; 胎儿期; 臀脂; 前体脂肪细胞; 原代分离培养; 成脂分化相关基因

Abstract: 【Objective】 The aim of this study was to isolate preadipocytes during the critical developmental phase of rump adipogenesis in Kazakh sheep,and investigate the developmental mechanism of rump adipose deposition in Kazakh sheep through histological and cytological analysis,so as to lay a foundation for investigating the molecular mechanisms underlying rump adipose deposition in sheep.【Method】 The embryos of Kazakh sheep at 50(E50),60(E60),70(E70),80(E80),94(E94),and 105(E105) days were collected,and the morphological characteristics of rump fat was observed.Oil Red O staining and adipogenic differentiation gene expression analysis were performed on rump adipose from embryos in Kazakh sheep during E70,E80,and E94 periods to determine the crucial phases of rump adipose deposition.Preadipocytes of rump adipose in Kazakh sheep during E70,E80,and E94 periods were isolated using a type Ⅰ collagenase stepwise digestion method.All three stages cells were subjected to 6 days continuous culture for growth curve plotting.Meanwhile,the proliferative capacity of preadipocytes from three embryonic stages was compared.Adipogenic induction and differentiation were performed on rump adipose preadipocytes in Kazakh sheep during E70,E80,and E94 periods.The differentiation potential was evaluated through Oil Red O staining,lipid droplet content quantification,and Real-time quantitative PCR analysis of adipogenic differentiation gene expression,and a comparative analysis of the adipogenic differentiation characteristics of preadipocytes across different embryonic stages was conducted.【Result】 There was obvious rump adipose in Kazakh sheep in E70 period,with visible lipid droplets initially forming rump at E80 period,followed by significant accumulation of lipid droplets in E94 period.Additionally,the expression of genes related to adipogenic differentiation gradually increased with gestation time.Notably,there was a fold increase from E80 to E94 periods.Furthermore，the precursor adipocytes at three developmental periods (E70,E80,and E94) from rump adipose isolated by type Ⅰ collagenase stepwise digestion method exhibited fibroblast-like with robust growth activity.Preadipocytes from all three periods were treated with induced differentiation to produce mature lipid droplets.The expression of adipogenic differentiation genes (CEBPα,PPARγ,LPL,and FABP4) in post-induction were significantly higher than that in pre-induction (P＜0.05).Compared with E70 or E94 periods,preadipocytes in E80 period had a higher capacity for lipid differentiation,earlier lipid droplet formation (3 day),and higher droplet content.【Conclusion】 The proliferation of rump adipose preadipocytes from isolated Kazakh sheep embryos during E70,E80 and E94 periods was well active.The cells could be induced to differentiate to form mature lipid droplets,and the differentiation ability of rump adipose preadipocyte was stronger in E80 period than that in the other two periods.

Key words: Kazakh sheep; fetal period; rump adipose; precursor adipocytes; primary isolation and culture; lipidogenic differentiation-related genes

中图分类号:

S826.8⁺4

张倩雯, 李婧平, 岳成广, 王嘉俊, 李忠慧, 李文蓉. 哈萨克羊胎儿期臀脂前体脂肪细胞的增殖和分化特性研究[J]. 中国畜牧兽医, 2025, 52(10): 4563-4574.

ZHANG Qianwen, LI Jingping, YUE Chengguang, WANG Jiajun, LI Zhonghui, LI Wenrong. Study on the Proliferation and Differentiation Characterization of Precursor Adipocytes from Rump Adipose in Kazakh Sheep During Fetal Period[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4563-4574.

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