鸡脂肪源间充质干细胞的分离与鉴定

发布时间：2018-11-15 19:01

【摘要】：【目的】建立鸡脂肪源间充质干细胞的分离与鉴定方法.【方法】用I型胶原酶消化法分离天露黄鸡脂肪间充质干细胞(AMSCs),CCK-8检测细胞生长活力,RT-PCR鉴定其特异性标记物,化学法对其进行成脂和成骨分化诱导.【结果和结论】原代及传代的细胞呈成纤维细胞样形态,并能传代至10代,其活力无明显变化;细胞生长曲线呈S型;RT-PCR检测显示AMSCs的特异性标志物CD71、CD44和CD29表达呈阳性,而属于造血干细胞的特异性标志物CD34和CD45呈阴性;AMSCs通过不同诱导液被成功诱导分化为成骨细胞和脂肪细胞,在成脂分化过程中有脂滴形成,油红O染色呈阳性,过氧化物酶体增殖物激活受体基因γ(PPARγ)和脂肪酸基因(FAS)的mRNA表达量升高;在成骨分化过程中有钙结节形成,茜素红染色呈阳性,碱性磷酸酶(ALP)活性检测对照组与诱导组比较差异显著(P㩳0.05),ALP基因和骨形态发生蛋白基因(BMP2)的mRNA表达量升高.研究表明,鸡AMSCs具有分化为多种细胞的潜能.
[Abstract]:[objective] to establish a method for isolation and identification of adipose derived mesenchymal stem cells from chicken. [methods] the (AMSCs), CCK-8 of adipose mesenchymal stem cells from Tianlu Yellow Chicken was isolated by type I collagenase digestion. RT-PCR identified its specific marker and induced its adipogenic and osteogenic differentiation by chemical method. [results and conclusion] the primary and passaged cells were fibroblast-like and could be subcultured to the 10th generation, but their activity did not change significantly. The expression of CD71,CD44 and CD29 in AMSCs was positive, but CD34 and CD45 in hematopoietic stem cells were negative. AMSCs was successfully induced to differentiate into osteoblasts and adipocytes through different induction fluids. Lipid droplets were formed in the process of adipogenic differentiation, and oil red O staining was positive. The mRNA expression of peroxisome proliferator-activated receptor gene 纬 (PPAR 纬) and fatty acid gene (FAS) was increased. Calcium nodules were formed during osteogenic differentiation and alizarin red staining was positive. The activity of alkaline phosphatase (ALP) in the control group was significantly different from that in the induced group (P0. 05). The mRNA expression of ALP gene and bone morphogenetic protein gene (BMP2) was increased. Studies have shown that chicken AMSCs has the potential to differentiate into a variety of cells.
【作者单位】：华南农业大学兽医学院;
【基金】：国家自然科学基金(31272519)
【分类号】：R329

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