生长分化因子5诱导脂肪干细胞复合Ⅰ型胶原支架成软骨细胞的分化

Chondrogenic differentiation of adipose-derived stem cells induced by growth differentiation factor-5 cultured on the type I collagen scaffold

背景:前期实验中发现生长分化因子5可以诱导脂肪干细胞向软骨细胞分化,但在复合Ⅰ型胶原支架的体外培养条件下其向软骨细胞分化的能力尚未见研究报道。目的:探讨生长分化因子5诱导脂肪干细胞复合Ⅰ型胶原支架向软骨细胞分化的能力。方法:从兔脂肪组织中分离培养脂肪干细胞,使用倒置相差显微镜观察细胞形态,使用免疫荧光对其表型进行鉴定。在复合Ⅰ型胶原支架条件下,加入外源性生长分化因子5对脂肪干细胞向软骨细胞进行诱导,在诱导14 d时,采用苏木精-伊红染色和扫描电镜对诱导的细胞进行形态学观察。在诱导7,14和21 d时,采用反转录PCR方法检测诱导的细胞的Ⅱ型胶原和蛋白多糖m RNA表达情况。结果与结论:原代脂肪干细胞贴壁生长,呈梭形、多角形分布,细胞表面抗原CD44、CD49d阳性,CD106阴性。生长分化因子5诱导的脂肪干细胞与Ⅰ型胶原支架黏附良好,增殖能力旺盛,细胞表面存在着大量的细胞外基质分泌,Ⅱ型胶原和蛋白聚糖m RNA表达水平明显增加。说明生长分化因子5能够成功诱导复合Ⅰ型胶原支架的脂肪干细胞成软骨细胞分化。

BACKGROUND： Growth differentiation factor-5 can induce adipose-derived stem cells into chondrocytes in our previous studies, but it has not been reported that the adipose-derived stem cells induced by growth differentiation factor-5 can differentiate into chondrocytes on the type I collagen scaffold. OBJECTIVE： To investigate the chondrogenic differentiation ability of adipose-derived stem cells induced by growth differentiation factor-5 cultured on the type I collagen scaffold. METHODS： Adipose derived stem cells were isolated from rabbit adipose tissue, the cells morphology was observed using inverted phase contrast microscope and the phenotypes were identified using immunofluorescence. The exogenous growth differentiation factor-5 was added to the cultural media with the type I collagen scaffold so as to induce the chondrogenic differentiation. The cells morphology was observed using hematoxyUn-eosin staining and scanning electron microscope after the induction by growth differentiation factor-5 for 14 days. Meanwhile, the type II collagen and aggrecan mRNA expressions of the induced ceils were measured using RT-PCR after the induction by growth differentiation factor-5 for 7, 14, and 21 days.RESULTS AND CONCLUSION： The primary cultured adipose-derived stem cells proliferated adherently with the fusiform and polygonal distribution under inverted phase contrast microscope. The positive CD44, CD49d and negative CD106 were detected by immunofluorescence. The adipose-derived stem cells induced by growth differentiation factor-5 were well adhered to the type I collagen scaffold and strongly proliferated. The large amounts of extracellular matrix existed on the surface of the induced cells under scanning electron microscope. RT-PCR agarose gel electrophoresis indicated that the type II collagen and aggrecan mRNA expressions of the adipose-derived stern cells induced by growth differentiation factor-5 with the type I collagen scaffold were significantly increased. Growth differentiation factor-5 can successfully induce the chondrogenic differentiation of adipose-derived stem cells cultured on the type I collagen scaffold.