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目的原代分离培养人真皮成纤维细胞(hDFs),应用生长分化因子-5(GDF-5)进行诱导培养,观察细胞的形态,检测软骨细胞相关指标,评价其分化为软骨样细胞的能力。方法取成人除皱术后的皮肤组织,Ⅰ型胶原酶消化法分离培养hDFs,观察细胞形态,MTT法检测细胞增殖活性,免疫细胞化学染色检测Ⅰ型胶原的表达。用100ng/ml GDF-5的成软骨诱导液培养,观察细胞形态变化,检测Ⅱ型胶原蛋白表达及分泌GAG的能力。结果 hDFs呈长梭形,P10以后仍保持较强的增殖潜能,Ⅰ型胶原表达阳性。诱导后的hDFs逐渐成为圆形、多角形,并聚集生长形成软骨样结节;诱导第21 d的细胞Ⅱ型胶原蛋白表达阳性,GAG被甲苯胺蓝染成蓝色。结论 hDFs经100ng/ml的GDF-5诱导培养可分化为软骨样细胞。
OBJECTIVE: Primary cultured human dermal fibroblasts (hDFs) were isolated and cultured, and induced by growth differentiation factor-5 (GDF-5). Morphological changes were observed. Chondrocyte-related indicators were assayed for their ability to differentiate into chondrocytes. Methods Human dermal tissues were obtained from human wrinkle removal. Type Ⅰ collagenase digestion was used to culture hDFs. Cell morphology was observed by MTT assay. Expression of collagen type Ⅰ was detected by immunocytochemical staining. Cultured with chondrocytes induced by 100ng / ml GDF-5, the morphological changes of cells were observed, and the expression of type Ⅱ collagen and the ability of secreting GAG were examined. Results The hDFs presented long fusiform shape and maintained a strong proliferative potential after P10. Type Ⅰ collagen expression was positive. The induced hDFs gradually became round, polygonal, and aggregated to form cartilage-like nodules. The expression of type II collagen in cells on day 21 was induced, and GAG was stained blue with toluidine blue. Conclusions hDFs can differentiate into chondrocytes after induced by 100ng / ml GDF-5.