CAJ | 학술논문

【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．
【목적】건립계지방원간충질간세포적분리여감정방법．【방법】용I형효원매소화법분리천로황계지방간충질간세포（AMSCs），CCK-8검측세포생장활력，RT-PCR감정기특이성표기물，화학법대기진행성지화성골분화유도．【결과화결론】원대급전대적세포정성섬유세포양형태，병능전대지10대，기활력무명현변화；세포생장곡선정S형；RT-PCR검측현시AMSCs적특이성표지물CD71、CD44화CD29표체정양성，이속우조혈간세포적특이성표지물CD34화CD45정음성；AMSCs통과불동유도액피성공유도분화위성골세포화지방세포，재성지분화과정중유지적형성，유홍O염색정양성，과양화물매체증식물격활수체기인γ（ PPARγ）화지방산기인（ FAS）적mRNA표체량승고；재성골분화과정중유개결절형성，천소홍염색정양성，감성린산매（ ALP）활성검측대조조여유도조비교차이현저（P﹤0.05），ALP기인화골형태발생단백기인（BMP2）적mRNA표체량승고．연구표명，계AMSCs구유분화위다충세포적잠능．
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 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 .