生长分化因子5诱导脂肪干细胞向软骨细胞的转化

Growth differentiation factor 5 induces adipose-derived stem cells differentiating into chondrocytes

背景:研究表明,生长分化因子5通过不同的调控机制在软骨发育中起到非常重要的作用。目的:观察生长分化因子5对脂肪干细胞分化的调控作用。方法:取第4代日本大耳白兔脂肪干细胞,分别以含0(空白对照组),10,50,100,150,200μg/L生长分化因子5的培养液培养,观察细胞形态变化,筛选出最佳生长分化因子5质量浓度。取最佳质量浓度生长分化因子5诱导培养1,2,3周的细胞爬片,分别进行免疫组织化学、甲苯胺蓝及免疫荧光染色。结果与结论:1最佳质量浓度:当生长分化因子5质量浓度为10,50μg/L时,细胞形态变化不明显;在200μg/L时,细胞呈现凋亡表现;在100,150μg/L质量浓度下,可见少量细胞由梭形变得不规则,部分细胞在7-10 d变成圆形或椭圆形,其中以100μg/L生长分化因子5诱导效果最好;2免疫组织化学染色:100μg/L生长分化因子5诱导后,转染细胞的Ⅱ型胶原化学染色显示细胞核蓝染,呈强阳性;3甲苯胺蓝染色:100μg/L生长分化因子5诱导后,转染细胞的细胞甲苯胺蓝染色阳性,胞浆异染;4免疫荧光染色:100μg/L生长分化因子5诱导组的蛋白聚糖绿色荧光表达逐渐增强,以诱导3周最为强烈;5结果表明:长分化因子5可促进脂肪干细胞向软骨细胞转化。

BACKGROUND：Growth differentiation factor 5 （GDF5） has been shown to play a crucial role in the development of chondrocytes via different regulatory mechanisms. OBJECTIVE：To explore the effect of GDF5 on the chondrogenic differentiation of adipose-derived stem cel s （ADSCs）. METHODS：ADSCs in passage 4 isolated from Japanese White rabbits were cultured in the medium containing 0 （blank control group）, 10, 50, 100, 150 and 200μg/L, respectively. The morphological changes were observed, and the optimal concentration of GDF-5 was screened, and its round coverslips at 1, 2 and 3 weeks of culture were selected to undergo immunocytochemistry, toluidine blue staining and immunofluorescent staining. RESULTS AND CONCLUSION：The growth of ADSCs was stable in the medium containing 10 and 50μg/L GDF5, while apoptotic cel s appeared after induction with 200μg/L GDF5. In the medium containing 100 and 150μg/L GDF5, some spindle-shaped cel s changed into irregular shape, and became round or oval after 7-10 days of culture. The optimal concentration of GDF5 was 100μg/L. The cel s transfected by 100μg/L GDF5 were positive for type II col agen obviously and with blue-stained nucleus. The transfected cel s were positive for toluidine blue, and metachromatic granules were visible in the cytoplasm. The proteoglycan mRNA expression of the transfected cel s was significantly increased, and reached the highest at 3 weeks. These results suggest that GDF5 promotes the chondrocytic differentiation of ADSCs.