犬脂肪干细胞的分离培养与鉴定  

Isolation,Culture and Identification of Canine Adipose-Derived Stem Cells

作　　者：黄奇[1] 周露云[1] 刘振兴[2] 王虹人[1] 施振声[1] 曾申明[2] 

机构地区：[1]中国农业大学动物医学院,北京海淀100193 [2]中国农业大学动物科技学院,北京海淀100193

出　　处：《中国兽医杂志》2014年第5期34-36,39,I0002,共5页Chinese Journal of Veterinary Medicine

摘　　要：为了研究脂肪干细胞（Adipose-derived stem cells,ASCs）体外培养方法及其生物学特性,将ASCs应用于小动物临床治疗,本试验用犬进行了脂肪干细胞的分离、培养和鉴定.犬脂肪干细胞（Canine adipose-derived stem cells,cASCs）体外培养生长良好,呈细长梭形.第3代（P3）细胞诱导2周后,成脂诱导组经油红O染色可见细胞内脂滴积累;成骨诱导组经碱性磷酸酶（Alkaline phosphatase,AKP）及VonKossa染色,AKP、钙结节均呈阳性表达.第3代至第5代（P3～P5）细胞流式细胞检测结果显示,所分离细胞稳定高表达间充质干细胞（Mesenchymal Stem Cells,MSCs）标志CD29、CD44、CD90（＞90％）,不表达造血细胞标志CD45（＜2％）.培养结果显示,从犬镰状韧带和皮下脂肪均可分离出cASCs.cASCs取材方便,易于体外培养,多次传代及冻存对cASCs的生物学性质无明显影响.cASCs有望成为国内动物医学临床研究和治疗的重要干细胞来源.To verify the method of ASCs culture in vitro and apply this method to the clinical therapy of veterinary medicine, ca- nine adipose tissue of 6 bitches was obtained in ovariohysterectomy for the isolation, culture and identification of, ASCs. ASCs grew well in vitro with spindle-like morphology. Passage 3 cells were analyzed for their capacity to differentiate into adipogenic and osteo- genie lineages. After 2 weeks, Oil Red 0 stain was positive in adipogenie differentiation medium, and AKP and yon Kossa stain were strongly positive in osteogenic differentiation medium. Flow cytometry showed that the majority of Passage 3 to Passage 6 of cASCs were positive for the mesenchymal markers CD29, CD44, CD90（〉90%）, and did not express the hematopoietie marker CD45（〈2%）. ASCs were successfully isolated from both falciform ligament and subcutaneous adipose tissue, and the cell proliferation may slow down with the increase of the age of a dog. In conclusion, cASCs were available and easy to culture, after several passages and the eryopres ervation period, the cells conserved their biological property, cASCs may represent an important stem cell source both for vet- erinary cell therapy and preclinical studies.

关 键 词：犬 脂肪干细胞 分离 鉴定 

分 类 号：S858.292[农业科学—临床兽医学]