绒山羊肌内前体脂肪细胞的基因表达分析

Gene Expression Analysis on Intramuscular Preadipocytes in the Cashmere Goats

本研究建立了山羊肌内前体脂肪细胞的体外培养模式,以期深入研究山羊肌内脂肪组织发育和脂肪沉积过程中标志基因的表达情况。试验采集1日龄羔羊背最长肌,Ⅱ型胶原酶消化1.5h后依次通过80目、400目筛网过滤,离心,通过差速贴壁法得到增殖旺盛肌内前体脂肪细胞,观察其形态学变化,油红O染色检测细胞内脂肪含量。实时定量PCR法检测LPL、PPAR、FTO、LIPIN基因的表达量。结果表明:原代培养的细胞成分均一,贴壁生长呈短梭状或棱形,具有肌内前体脂肪细胞的形态特征。通过定量分析,FTO mRNA在早期就有较高表达,在分化5~7d达到最高水平,之后维持较高水平,11d表达量显著下降(P<0.05)。LIPIN mRNA在加入诱导剂后出现波动现象。PPARmRNA在整个细胞分化和增殖过程中维持一个较稳定的水平。LPLmRNA在分化早期高表达,随着分化程度的增加表达量逐渐降低。本研究成功建立了山羊肌内前体脂肪细胞原代培养方法,并在体外重现了增殖和分化的过程。基因表达量为FTO>LPL>PPAR>LIPIN,这可为进一步研究脂肪沉积机制及优化山羊肉品品质奠定基础。

The intramuscular preadipocytes culture model in vitro of goat was established in this study, in order to research marker gene expression in intramuscular adipose tissue development and fat deposition in goat. The longissimus muscle of 1-day-old lamb was collected in this experi- ment, and digested by type II collagenase for 1.5 h, then filtered by 80 mesh, 400 mesh in se quence, after centrifugation, proliferative intramuscular preadipocytes were obtained using differ- ential adhesion method. The morphological changes were observed, and fat content in cells was detected by oil red O staining. The genes expression of LPL, PPAR, FTO and LIPIN was de- tected using real time quantitative PCR. The results showed that. the primary cultured cells were homogeneous, and the shortening-shaped or prismatic cells were showed in adherent growth,with the morphological characteristics of the intramuscular preadipocytes. The mRNA of FTO gene had a higher expression level in early stage, and the peak occurred at the 5-7 d in differentia- tion, and thereafter maintained a high level, then expression amount significantly decreased till 11 d （P^0.05）. LIPIN mRNA fluctuated after the addition of inducer. PPAR mRNA main- tained a relatively stable level in whole differentiation and proliferation process. LPL mRNA was expressed highly in the early stages of differentiation, and the expression decreased gradually with the increase of the degree of differentiation. The primary culture method of intramuscular preadipocytes in goat was established successfully in this research, and proliferation and differen- tiation process were reproduced in vitro. The amount of gene expression by quantitative analysis was： FTO〉 LPL〉 PPAR〉 LIPIN. This research laided the basis for further study of the fat deposition mechanism of intramuscular adipose tissue and improving the quality of goat.