小尾寒羊前体脂肪细胞分化过程相关基因表达规律的研究

doi:10.16431/j.cnki.1671-7236.2019.07.018

摘要/Abstract

摘要：

为了探究小尾寒羊脂肪细胞分化过程中相关基因的变化规律，试验采集2月龄小尾寒羊腹股沟白色脂肪组织，通过酶消化法体外分离小尾寒羊前体脂肪细胞。培养前体脂肪细胞布满细胞板后，分别用诱导Ⅰ液、诱导Ⅱ液对细胞进行诱导分化，使其成为成熟的脂肪细胞。利用油红O染色法验证成熟脂肪细胞并检测脂滴含量。分别在增殖期细胞增殖70%、90%及分化期诱导Ⅰ液处理48 h、诱导Ⅱ液处理48 h、完全培养液处理48 h时（2、4、6、8、10 d）提取细胞总RNA，反转录成cDNA。采用实时荧光定量PCR检测PPARγ、C/EBPα、LPL、SREBP1、KLF5、KLF6、FABP4、STAT5、ACSS2、IGF1、ADD1、FOXO1、ACACA、DGAT1、CPT1A基因的表达规律。结果表明，试验成功分离并诱导前体脂肪细胞变为成熟的脂肪细胞，细胞内部具有明显脂滴；实时荧光定量PCR结果表明，上述基因在细胞分化阶段具有明显波动，峰值出现的时间均不相同；C/EBPα、FOXO1基因表达峰值出现在第6天，可能在细胞分化早期发挥作用；PPARγ、LPL、SREBP1、KLF5、KLF6、FABP4、STAT5、ADD1、ACSS2基因表达峰值出现在第8天，但表达倍数与趋势均不相同；ACACA基因表达量出现上下波动；IGF1、DGAT1基因表达峰值出现在第10天；CPT1A基因表达量则一直下降；FABP4基因表达倍数显著高于其他基因。本研究全面检测了小尾寒羊前体脂肪细胞在分化过程中关键基因的表达规律，可为探究小尾寒羊脂肪分化过程分子机制、挖掘参与脂肪分化新的关键基因、提高小尾寒羊肌间脂肪含量等研究提供一定的理论参考。

关键词: 小尾寒羊; 前体脂肪细胞; 分离培养; 分化; 脂肪细胞; 基因表达

Abstract:

In order to explore the changes of related genes during adipocyte differentiation of Small-tail Han sheep,white inguinal adipose tissuees of two-month-old Small-tail Han sheep were collected and preadipocytes were isolated by enzymatic digestion in vitro.After the cultured preadipocytes were covered with cell plates,the cells were induced to differentiate into mature adipocytes by induction solution Ⅰ and induction solution Ⅱ,respectively.Oil red O staining was used to verify adipocytes and detect lipid droplets.Total RNA was extracted from 70% and 90% of the proliferative cells,48 h of induction Ⅰ,48 h of induction Ⅱ and 48 h of complete culture medium (2,4,6,8,10 d,respectively),and was retranscribed into DNA.Real-time quantitative PCR (RT-qPCR) was used to detect genes PPARγ,C/EBPα,LPL,SREBP1,KLF5,KLF6,FABP4,STAT5,ACSS2,IGF1,ADD1,FOXO1,ACACA,DGAT1 and CPT1A that had been shown to be involved in the differentiation of human and mouse preadipocytes to explore their expression in the differentiation of Small-tail Han sheep.The results showed that the preadipocytes were successfully separated and induced to become mature adipocytes with obvious lipid droplets inside the cells;RT-qPCR assay showed that the expressions of above genes had significant fluctuations in the cell differentiation stage,and the peak time was different;The peak expression of C/EBPα and FOXO1 appeared on the 6th day,which may play an important role in the early stage of cell differentiation.PPARγ,LPL,SREBP1,KLF5,KLF6,FABP4,STAT5,ADD1 and ACSS2 gene peaks appeared on the 8th day,but the expression multiples and trends were different;The expression of ACACA gene fluctuated up and down.The peaks of IGF1 and DGAT1 appeared on the 10th day.The expression of CPT1A was declining;The expression multiple of FABP4 gene was significantly higher than that of other genes.This study completely detected the expression regulation of key genes in the differentiation process of Small-tail Han sheep preadipocytes.The results could provide theoretical reference for exploring the molecular mechanism of Small-tail Han sheep adipose differentiation,discovering new key genes involved in adipose differentiation and improving the intermuscular fat content of Small-tail Han sheep.

Key words: Small-tail Han sheep; preadipocytes; isolation and culture; differentiation; adipocytes; gene expression

中图分类号:

S811.3

肖成, 金海国, 魏天, 曹阳. 小尾寒羊前体脂肪细胞分化过程相关基因表达规律的研究[J]. 《中国畜牧兽医》, 2019, 46(7): 2030-2037.

XIAO Cheng, JIN Haiguo, WEI Tian, CAO Yang. Study on the Expression of Genes Related to the Differentiation Process of Preadipocytes in Small-tail Han Sheep[J]. , 2019, 46(7): 2030-2037.

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