摘要
研究旨在建立滩羊肌内前体脂肪细胞的体外培养体系,并探讨其在诱导分化过程中成脂关键基因的表达规律。采集10日龄滩羊背最长肌组织,用Ⅱ型胶原酶消化分离肌内前体脂肪细胞,进行细胞的原代、传代培养和形态学观察,继而对获得的细胞进行诱导分化和油红O染色鉴定。同时,采用qRT-PCR检测参与前体脂肪细胞成脂分化的关键基因PPARγ、C/EBPα、FAS、aP2、LPL mRNA在诱导第0、2、4、6、8 d的表达水平变化。结果表明:(1)滩羊原代肌内前体脂肪细胞在分离后约4 h时开始贴壁,贴壁的细胞呈现为小梭形与不规则三角形。经过传代后,细胞成分均一并且贴壁良好,呈现成纤维细胞样形态;细胞传代第2~6 d为指数生长期,其生长曲线近似“S”形;(2)细胞诱导分化第8天时,油红O染色呈红色,细胞具有前体脂肪细胞的典型特征;(3)PPARγ和FAS的表达水平在第6天时最高,随着诱导时间增长PPARγ表达先升高后降低,FAS表达先降低后升高再降低;C/EBPα、aP2和LPL的表达量在第4 d最高,随着诱导分化时间增长,C/EBPα和LPL表达先升高后降低,aP2表达先升高后降低再升高。
The aim of this study was to establish a culture system for intramuscular preadipocytes in Tan sheep in vitro,and to explore the expression rules of key genes for adipogenesis during differentiation.The longissimus muscle of 10-day-old Tan sheep was collected and digested with collagenase typeⅡto separate the intramuscular preadipocytes for primary and subculture culture and morphological observation.The obtained cells were subjected to induced differentiation and oil red O staining identification.At the same time,qRT-PCR was used to detect the expression levels of the key genes PPARγ,C/EBPα,FAS,aP2,and LPL mRNA related to adipogenic differentiation of intramuscular adipocytes on the 0th,2nd,4th,6th,and 8th days of induction.Results showed:(1)primary intramuscular preadipocytes began to adhere at about 4 h after separation,and the adherent cells appeared as fusiform and irregular triangles;after subculture,the cell components were uniform and adhered well,showing fibroblast-like morphology;the second to sixth days of cell subculture were at the exponential growth phase,and its growth curve was similar to"S"shape;(2)on the 8th day of differentiation,the oil red O stained red,and the cells had the typical characteristics of preadipocytes;(3)the expression levels of PPARγand FAS were the highest on the 6th day;as the induction time increased,the expression of PPARγincreased first and then decreased,and the expression of FAS decreased first and then increased and then decreased;the mRNA expression of C/EBPα,aP2 and LPL was the highest on the 4th day;as the induction time increased,the expression of C/EBPαand LPL increased first and then decreased,and the expression of aP2 increased first and then decreased and then increased.
作者
徐小春
陈文娟
赵瑞
马森
XU Xiaochun;CHEN Wenjuan;ZHAO Rui;MA Sen(College of Biological Science and Engineering,North Minzu University,Yinchuan,Ningxia 750021,China;College of Animal Science and Technology,Henan Agricultural University,Zhengzhou,Henan 450002,China)
出处
《家畜生态学报》
北大核心
2020年第11期35-41,共7页
Journal of Domestic Animal Ecology
关键词
滩羊
肌内前体脂肪细胞
诱导分化
成脂基因
Tan sheep
intramuscular preadipocytes
induced differentiation
adipogenic genes
