兔脂肪间充质干细胞分离培养及鉴定

Isolation,Culture and Characterization of Rabbit Adipose-derived Stromal Cells

为了建立一种体外培养兔脂肪间充质干细胞(ADSCs)的方法,在无菌条件下取家兔双侧腹股沟脂肪垫,Ⅰ型胶原酶消化后,用含10%胎牛血清的DMEM培养液培养。细胞连续传代后,MTT法检测第3、5、10代细胞的增殖情况。采用油红染色法和Von Kossa染色法观察第3代ADSCs向成脂和成骨方向分化情况,并采用流式细胞仪分析细胞表面标志。结果显示,第3、5、10代的ADSCs生长曲线并无显著差异,细胞增殖较快;ADSCs成脂诱导后油红染色阳性,成骨诱导后Von kossa染色形成典型的黑色钙化结节。ADSCs表面标志检测结果为:CD29和CD90阳性,CD45和CD80阴性。成功分离和培养了兔的ADSCs,其具有成脂和成骨潜能,可作为组织工程的种子细胞。

In order to establish a method for isolation and culture of rabbit adipose -derived stromal ceils in vitro and explore their biological characteristics. Inguinal fat pads of rabbit were aseptically separated, followed by digestion with I type collagenase, then cultured in DMEM with 10% fetal bovine serum and assay its growth curve of the third, fifth and tenth generation. To verify the multipotential differentiation of mesenchymal characteristics of ADSCs, the third passage cells were subjected to differentiation in conditions known to induce adipogenic and osteogenic lineages and used for phenotypic analysis by flow cytometry. There were no significant deviation between growth curve of the third, fifth and tenth passage. Cells had a strong capability of proliferation. After cultured in adipogenie differentiation medium, the cells were Oil Red O staining positive. After cultured in osteogenic differentiation medium, the cells were Von kossa staining positive. The cultured ADSCs in this study were positive for CD29, CD90 and negative for CD80 and CD45. ADSCs were successfully isolated and cultured from rabbit adipose tissue. These cells have adipogenic and osteogenic potential. This study showed that the ADSCs could be served as seed cells in tissue engineering.