延黄牛前体脂肪细胞的培养及生物学特性研究

Primary Culture and Biological Characteristics of Preadipocytes in Yanhuang Cattle

近几年来延黄牛因肉质优良、口味独特、有典型的大理石花纹等特点入选为我国五大良种牛之一,越来越多地受到消费者的青睐。为建立延黄牛前体脂肪细胞的体外培养体系,从细胞和分子水平探索延黄牛脂肪细胞的生物学特性及潜在的分化机制,采用胶原酶消化法对延黄牛皮下前体脂肪细胞进行分离,对其进行形态学观察并绘制生长曲线,油红O染色法检测成脂诱导分化过程中的脂质积累,利用甘油三酯酶法测定细胞内甘油三酯,采用实时荧光定量RT-PCR的方法检测脂肪细胞成脂标志基因过氧化物酶体增殖体激活受体γ(PPARγ)、CCAAT/增强子结合蛋白α(C/EBPα)的mRNA在延黄牛前体脂肪细胞分化过程中的表达。结果表明:经过分离的延黄牛原代脂肪细胞可贴壁生长,传代后成分均一贴壁良好,呈带角的梭型、形似成纤维细胞;第2天开始细胞进入对数生长期,第4天以后为细胞生长的平台期,增殖速度明显变慢,细胞生长曲线近视"S"形,符合正常的细胞生长规律;经诱导分化后的细胞可被油红O染色,呈现典型的圆形脂滴,具有成熟脂肪细胞的典型特征;甘油三酯含量在第0~4天变化缓慢,在第4天以后显著升高;成脂标志基因PPARγ、C/EBPα随着分化过程的进行,在分化早期显著上升,随着分化进程的结束略有下降。综上所述,本研究成功建立了延黄牛前体脂肪细胞的分离培养方法,为进一步研究延黄牛脂肪沉积作用机制以及改善牛肉品质奠定了基础。

In recent years,Yanhuang cattle,one of the Chinese five major breeds of cattle,has been received more and more attention because of its excellent meat quality,unique taste,and typical characteristics of the marble pattern.The aim of this study was to establish the preadipocyte in vitro cultures system and explore the biological characteristics and potential mechanism of Yanhuang cattle adipocyte at the cellular and molecular level,and lay a foundation for improvement of beef quality.The subcutaneous adipose tissue of bovine was isolated by collagenase digestion method,and the morphological characteristics were observed;the growth curve was drawn by MTS method;the lipid accumulation was detected by oil red O staining;the triglyceride method was used to detected intracellular triglyceride content;andreal-time fluorescent quantitative RT-PCR was used to detect the expression of adipogenic marker genes including peroxisome proliferator activated receptor(PPARγ)and CCAAT/enhancer binding proteinα(C/EBPα)mRNA expression during the differentiation of buffalo preadipocytes.The results showed that the isolated adipocytes of primary adipocytes from Yanhuang cattle presented monolayer growth,the components of preadipocytes were uniform with the shape of fibroblasts after passage.The cells began to logarithmic growth phase from the second day,and reached the plateau phase after the fourth day,with obviously slow proliferation rate.The cell growth curve was approximately"S"shape,which was accorded with the normal law of cell growth.The differentiated cells could be stained with Oil Red O,showing the typical round lipid drop lets with the typical characteristics of mature adipocytes.The content of triglyceride changed slowly from Day 0 to Day 4 and significantly increased after Day 4.The adipogenic marker gene PPAR酌,C/EBP琢significantly increased in the early stage of differentiation as the differentiation progressed,and decreased slightly with the end of the differentiation process.In summary,the present study has successfully established the method of isolation and culture of Yanhuang cattle preadipocytes,which lays a foundation for further study on the mechanism of fat deposition in Yanhuang cattle and the improvement of beef quality.