【作者】 唐小洪； 马浩然； 叶亚琼； 马勇江； 张媛； 李玉谷；

【Author】 TANG Xiaohong;MA Haoran;YE Yaqiong;MA Yongjiang;ZHANG Yuan;LI Yugu;College of Veterinary Medicine,South China Agricultural University;

【机构】 华南农业大学兽医学院；

【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。【结果与结论】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γ(PPARγ)和脂肪酸(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)活性显著升高(P<0.05),ALP和骨形态发生蛋白(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。更多还原

【Abstract】 [Objective]A method for isolation and identification of chicken adipose-derived mesenchymal stem cells(AMSCs)was established.[Method]AMSCs from Tianlu yellow chicken were obtained by type Ⅰ collagenase digestion.CCK-8 was used to detect cell activity,RT-PCR was used to examine their specific molecular makers,and their adipogenic and osteogenic differentiation were chemically induced.[Result and conclusion]The primary cultured and subcultured cells showed fibroblast-like morphology,and primary AMSCs were subcultured to passage 10 without any change in activities.The growth curves were typically sigmoidal.RT-PCR assay showed that the specific makers of AMSCs,CD29,CD44 and CD71,were positive,but CD34 and CD45 characterized by hematopoietic stem cells were negative.In addition,AMSCs can successfully differentiate into osteoblasts and adipocytes in different media.Lipid droplets formation was recorded during adipogenic induction,the cells were positive for oil red O staining,and mRNA expression of peroxisome proliferator-activated receptor-γ(PPARγ) and fatty acid(FAS) was increased.During osteogenic induction,alizarin red staining showed that the calcium nodus was positive,the alkaline phosphatase(ALP) activity is higher in the test group than control group(P < 0.05),mRNA expression of ALP and bone morphogenetic protein-2(BMP2) was increased.This study suggests that the mesenchymal stem cells isolated from chicken adipose tissue are multi-potential and may provide the possibility for future clinical application.更多还原