【摘要】：本試驗旨在建立梅山豬前體脂肪細胞的體外培養(yǎng)體系,從細胞和分子水平探索梅山豬脂肪組織增生的生物學特征。采集3日齡梅山豬仔豬頸、背部皮下脂肪組織,采用Ⅰ型膠原酶分離前體脂肪細胞,進行前體脂肪細胞的原代和傳代培養(yǎng),并對其進行形態(tài)學觀察、生長曲線測定、油紅O染色鑒定及脂肪含量的測定,且采用實時熒光定量RT-PCR的方法檢測脂肪細胞分化關(guān)鍵基因PPARγ、CEBPα、LPL、DLK1、FABP4及ADIPOQ mRNA在梅山豬前體脂肪細胞分化過程中的表達。結(jié)果表明:分離的梅山豬原代前體脂肪細胞約6 h開始貼壁,貼壁的細胞呈小梭形或不規(guī)則的三角形,經(jīng)傳代后成分均一,貼壁良好,呈成纖維細胞樣形態(tài),第1~9天細胞進入指數(shù)生長期,細胞生長曲線近似S形;誘導培養(yǎng)第11天細胞充脂率高,油紅O染色呈紅色,細胞內(nèi)脂肪含量顯著高于未誘導組,具有前體脂肪細胞的典型特征;PPARγ在豬前體脂肪細胞分化早期就有表達,分化第3天高度表達,CEBPα和LPL隨著分化過程表達量逐漸增加,而DLK1表達量逐漸下降,FABP4和ADIPOQ在第6天高度表達。綜上,本研究成功建立了梅山豬前脂肪細胞的培養(yǎng)方法和誘導分化方法,為進一步研究梅山豬體脂沉積和改善肉品質(zhì)奠定基礎(chǔ)。
[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 緯, CEBP 偽, 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 緯 was expressed at the early stage of porcine preadipocyte differentiation. On the 3rd day of differentiation, the expression of CEBP 偽 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.
【作者單位】： 南京農(nóng)業(yè)大學動物科技學院;
【基金】：國家自然科學基金(31501930) 中央高�；究蒲袠I(yè)務(wù)費(KJQN201606) 江蘇省自然科學基金(BK20150656)
【分類號】：S828

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