摘要
本试验旨在建立梅山猪前体脂肪细胞的体外培养体系,从细胞和分子水平探索梅山猪脂肪组织增生的生物学特征。采集3日龄梅山猪仔猪颈、背部皮下脂肪组织,采用Ⅰ型胶原酶分离前体脂肪细胞,进行前体脂肪细胞的原代和传代培养,并对其进行形态学观察、生长曲线测定、油红O染色鉴定及脂肪含量的测定,且采用实时荧光定量RT-PCR的方法检测脂肪细胞分化关键基因PPARγ、CEBPα、LPL、DLK1、FABP4及ADIPOQ mRNA在梅山猪前体脂肪细胞分化过程中的表达。结果表明:分离的梅山猪原代前体脂肪细胞约6 h开始贴壁,贴壁的细胞呈小梭形或不规则的三角形,经传代后成分均一,贴壁良好,呈成纤维细胞样形态,第1~9天细胞进入指数生长期,细胞生长曲线近似S形;诱导培养第11天细胞充脂率高,油红O染色呈红色,细胞内脂肪含量显著高于未诱导组,具有前体脂肪细胞的典型特征;PPARγ在猪前体脂肪细胞分化早期就有表达,分化第3天高度表达,CEBPα和LPL随着分化过程表达量逐渐增加,而DLK1表达量逐渐下降,FABP4和ADIPOQ在第6天高度表达。综上,本研究成功建立了梅山猪前脂肪细胞的培养方法和诱导分化方法,为进一步研究梅山猪体脂沉积和改善肉品质奠定基础。
The aim of this study was to establish the preadipocyte in vitro culture system and understand the biological characteristics of expansion of Meishan pig adipocyte tissue at the cellular and molecular level. Preadipocytes were obtained from subcutaneous adipocyte tissue of neck and back from 3-day Meishan pigs by digestion of type I collagenase,then primary cultured and sub-cultured. Morphological change,growth curve,Oil red O staining and fat content were studied,and mRNA expressions of PPARγ,CEBPα,LPL,DLK1,FABP4 and ADIPOQ measured during Meishan pig preadipocyte differentiation by real-time fluorescent quantitative RT-PCR. The results showed that Meishan piglet primary preadipocytes began to paste the wall about 6 hours and presented small spindle-shape and irregular triangle. The cells were highly homogeneous,adhered well and presented fibroblast-like morphology after sub-culturing. During 1-9 days,the cells entered logarithmic growth phase and growth curve showed ＂S＂shape. At 11 day of induced culture,the cells had a high differentiation rate and showed red by Oil red O staining. Fat content was exceeded significantly than the un-induced groups. Thus,the cells presented typical characteristics of preadipocytes. PPARγ gene expressed in the early stage of preadipocyte differentiation and highly expressed on 3rd day. CEBPαand LPL gene levels gradually increased with preadipocyte differentiation,while DLK1 gene levels gradually decreased. FABP4 and ADIPOQ gene highly expressed on 6th day. In all,this study established a culture system and induced differentiation model for Meishan pigs successfully,providing a solid basis for further studying fat deposition and pork quality in Meishan pigs.
出处
《畜牧与兽医》
北大核心
2017年第6期9-14,共6页
Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31501930)
中央高校基本科研业务费(KJQN201606)
江苏省自然科学基金(BK20150656)
关键词
梅山猪
前体脂肪细胞
细胞培养
诱导分化
Meishan piglets
preadipocyte
cell culture
induced differentiation
