猪肌源性前体脂肪细胞的分离培养和成脂诱导分化研究

doi:10.11843/j.issn.0366-6964.2018.12.010

摘要/Abstract

摘要：

本研究旨在建立猪肌源性前体脂肪细胞的体外培养体系，并探究成脂诱导分化过程中相关基因的表达，为进一步研究调控猪肌内脂肪沉积的分子机制提供试验材料。采集15日龄仔猪的背最长肌组织，使用Ⅱ型胶原酶进行消化，分离获得前体脂肪细胞，进行原代、传代培养以及成脂诱导，观察细胞形态、测定生长曲线、油红O染色鉴定以及检测成脂关键基因PPARγ、FABP4、C/EBPα、C/EBPβ、ZFP423、SREBP1、PRDM16和HSL在诱导分化过程中的表达量。结果表明，分离的猪肌源性前体脂肪细胞在6~7 h，发现少量细胞开始贴壁，贴壁的细胞呈似圆型、小梭型以及其它不规则形状，经传代后细胞形态均一，呈典型的成纤维细胞样，细胞的生长曲线呈S型。添加分化培养基进行诱导后，所有基因在细胞分化的早期均有表达，FABP4、C/EBPα、SREBP1与PPARγ基因在分化早期表达量极显著升高（P<0.01），在后期表达量极显著下降（P<0.01），C/EBPβ、ZFP423和PRMD16基因的表达量在分化过程中未见显著变化，HSL的表达量随着分化进行极显著降低（P<0.01）。在诱导分化12 d左右，油红O染色呈现红色。综上表明，本研究成功建立了猪肌源性前体脂肪细胞的分离与培养体系，进行成脂诱导分化，揭示分化过程中关键基因的表达规律，为进一步研究猪肌内脂肪沉积的分子机制和改善肉质品质奠定基础。

Abstract:

The aim of this study was to establish the culture system of porcine myogenic adipocytes cell in vitro, and to understand the mRNA expression regulation of adipogenesis related genes during adipogenic differentiation process and to provide experimental materials for further studying on the mechanism of intramuscular fat deposition in pig. Samples of longissimus dorsi muscle of the 15 days old piglets were collected and the precursor adipocytes cell was obtained by digested with type Ⅱ collagenase, and the cell were separated, primary and passage cultured and adipogenic induction. The morphology of cell were observed and the growth curve was determined. The adipogenesis was measured by Oil red O staining and the mRNA expression of adipogenesis related genes, such as PPARγ, FABP4, C/EBPα, C/EBPβ, ZFP423, SREBP1, PRMD16 and HSL were detected by qRT-PCR during induced differentiation. The results showed that a small number of myogenic preadipocytes cells began to adhere to the cell wall at about 6-7 h, and the shape of the cells were round, small shuttle and irregular, and became uniform in fibroblast-like appearance after cell passage. The growth curve was typical "S" shaped. All genes detected in this study were expressed at early stage of adipogenic differentiation after adding differentiation medium, in which the mRNA expression of FABP4, C/EBPα, SREBP1 and PPARγ at early stage was very significantly higher (P<0.01) and then was decreased significantly (P<0.01). There was no significant change for the mRNA expression of C/EBPβ,ZFP423 and PRMD16 genes during different differentiation stages. The mRNA expression of HSL was significantly reduced with the differentiation process (P<0.01). Oil red O staining was red after induction for 12 days. In conclusion,The system of the isolation, culture and adipogenic differentiation of porcine myogenic adipocytes cell was successfully established, and the expression and regulation of key genes involved in adipogenesis was investigated during cell differentiation process, which provide the foundation for further studying on the mechanism of intramuscular fat deposition and improvement of meat quality in pig production.

中图分类号:

S828.2

张宁芳, 成志敏, 乐宝玉, 秦本源, 王媛媛, 王鹤洁, 高鹏飞, 郭晓红, 李步高, 曹果清. 猪肌源性前体脂肪细胞的分离培养和成脂诱导分化研究[J]. 畜牧兽医学报, 2018, 49(12): 2612-2621.

ZHANG Ning-fang, CHENG Zhi-min, LE Bao-yu, QIN Ben-yuan, WANG Yuan-yuan, WANG He-jie, GAO Peng-fei, GUO Xiao-hong, LI Bu-gao, CAO Guo-qing. Isolation, Culture and Adipogenic Differention of Pig Myogenic Preadipocytes Cell[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(12): 2612-2621.

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https://www.xmsyxb.com/CN/Y2018/V49/I12/2612

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