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目的分离培养人脂肪干细胞(hADSCs),用不同浓度GDF-5进行诱导,观察细胞形态变化及检测相关蛋白的表达,评价其是否具有软骨样细胞的特性。方法取人抽脂术后的无菌脂肪组织,酶消化法分离培养hADSCs,观察细胞形态,MTT法检测细胞增殖能力。用含0、50、100ng/mlGDF-5的软骨诱导液培养,观察形态变化,检测Ⅱ型胶原及GAG的表达。结果 hADSCs呈长梭形,P10以后仍具有较强的增殖能力。经GDF-5诱导的hADSCs逐渐变为圆形、多角形,部分细胞聚集生长,形成软骨样结节;诱导28d的细胞Ⅱ型胶原表达阳性,甲苯胺蓝将GAG染成蓝色,但100ng/mlGDF-5诱导组阳性表达明显高于50ng/ml诱导组。结论 hADSCs在100ng/mlGDF-5的诱导下可分化为软骨样细胞。
OBJECTIVE: To isolate and culture human adipose-derived stem cells (hADSCs) and induce them with different concentrations of GDF-5 to observe the changes of cell morphology and the expression of related proteins, and to evaluate whether they possess the characteristics of cartilage-like cells. Methods Adipose tissue was obtained after liposuction. The hADSCs were isolated and cultured by enzymatic digestion. The cell morphology was observed. The cell proliferation was detected by MTT assay. Cultured with cartilage containing 0, 50 and 100 ng / ml GDF-5, the morphological changes were observed and the expression of type Ⅱ collagen and GAG were detected. HADSCs showed a long spindle shape, P10 still has a strong ability to proliferate. After induced by GDF-5, hADSCs gradually became round and polygonal, some cells aggregated and grew to form cartilage-like nodules. The expression of type Ⅱ collagen was observed on day 28 after induction with toluidine blue. However, 100ng / mlGDF-5 induced group was significantly higher than the 50ng / ml induction group. Conclusion hADSCs can differentiate into chondrocytes under the induction of 100ng / ml GDF-5.