鸡脂肪源间充质干细胞的分离与鉴定

Isolation and identification of chicken adipose-derived mesenchymal stem cells

【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﹤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.

Objective A method for isolation and identification of chicken adipose-derived mesenchymal stem cells（ AMSCs） was established .[Method] AMSCs from Tianlu yellow chickens were obtained by type I collagenase digestion .CCK-8 was used to detect cell activities .RT-PCR was used to examine their specific marker . Whereas their adipogenic and osteogenic differentiations 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 assays showed that the specific markers of AMSCs , CD29, CD44 and CD71, were positive , but CD34 and CD45 characterized by hematopoietic stem cells were negative .In addition , AMSCs can successfully differentiated into osteoblasts and adipocytes in different media .Lipid droplets formation was recorded during adipogenic induction , with the cells being 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, and there was significant difference in alkaline phosphatase （ALP） activities between the＆nbsp;test and control group （ P 〈0.05 ） , mRNA expression of ALP and bone morphogenetic protein-2 （ BMP2 ） also increased .This research suggests that the mesenchymal stem cells isolated from chicken adipose tissues are multi-potential and may provide the possibility for future clinical application .