梅山猪前体脂肪细胞的分离培养及分化相关基因的表达规律研究
发布时间：2019-02-14 20:49
【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。
[Abstract]:The aim of this study was to establish a culture system of preadipocytes from Meishan pig in vitro, and to explore the biological characteristics of adipose tissue proliferation in Meishan pig at cellular and molecular level. The subcutaneous adipose tissue of the neck and back of Meishan piglets at the age of 3 days was collected and the precursor adipocytes were isolated by type I collagenase. The primary and passage culture of the preadipocytes was carried out. The morphology and growth curve of the cells were observed and measured. Oil red O staining and fat content determination were used to detect the expression of PPAR 纬, CEBP 伪, LPL,DLK1,FABP4 and ADIPOQ mRNA in the differentiation of adipocyte precursor cells in Meishan pig by real-time fluorescence quantitative RT-PCR. The results showed that the primary preadipocytes of Meishan pig began to adhere to the wall for about 6 h. The adherent cells were small fusiform or irregular triangle. After passage, they were homogeneous in composition, good in adherent to the wall and in fibroblast-like shape. On the 1st day, the cells entered the exponential growth stage, and the cell growth curve was similar to S shape. On the 11th day after induction, the fat-filling rate of the cells was high, the oil red O staining was red, and the fat content in the cells was significantly higher than that in the uninduced group, which had the typical characteristics of the precursor adipocytes. PPAR 纬 was expressed at the early stage of porcine preadipocyte differentiation. On the 3rd day of differentiation, the expression of CEBP 伪 and LPL increased gradually, while the expression of DLK1 decreased. FABP4 and ADIPOQ were highly expressed on the 6th day. In conclusion, the method of culture and differentiation of preadipocytes in Meishan pig was established successfully, which laid a foundation for further study on fat deposition and meat quality improvement in Meishan pig.
【作者单位】： 南京农业大学动物科技学院;
【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)
【分类号】：S828
[Abstract]:The aim of this study was to establish a culture system of preadipocytes from Meishan pig in vitro, and to explore the biological characteristics of adipose tissue proliferation in Meishan pig at cellular and molecular level. The subcutaneous adipose tissue of the neck and back of Meishan piglets at the age of 3 days was collected and the precursor adipocytes were isolated by type I collagenase. The primary and passage culture of the preadipocytes was carried out. The morphology and growth curve of the cells were observed and measured. Oil red O staining and fat content determination were used to detect the expression of PPAR 纬, CEBP 伪, LPL,DLK1,FABP4 and ADIPOQ mRNA in the differentiation of adipocyte precursor cells in Meishan pig by real-time fluorescence quantitative RT-PCR. The results showed that the primary preadipocytes of Meishan pig began to adhere to the wall for about 6 h. The adherent cells were small fusiform or irregular triangle. After passage, they were homogeneous in composition, good in adherent to the wall and in fibroblast-like shape. On the 1st day, the cells entered the exponential growth stage, and the cell growth curve was similar to S shape. On the 11th day after induction, the fat-filling rate of the cells was high, the oil red O staining was red, and the fat content in the cells was significantly higher than that in the uninduced group, which had the typical characteristics of the precursor adipocytes. PPAR 纬 was expressed at the early stage of porcine preadipocyte differentiation. On the 3rd day of differentiation, the expression of CEBP 伪 and LPL increased gradually, while the expression of DLK1 decreased. FABP4 and ADIPOQ were highly expressed on the 6th day. In conclusion, the method of culture and differentiation of preadipocytes in Meishan pig was established successfully, which laid a foundation for further study on fat deposition and meat quality improvement in Meishan pig.
【作者单位】： 南京农业大学动物科技学院;
【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)
【分类号】：S828
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