GDF-5在体外诱导脂肪干细胞向软骨细胞转化实验研究

Experimental study on induction of adipose-derived stem cells into chondrocytes by GDF-5 in vitro

目的研究体外生长分化因子5(GDF-5)对脂肪干细胞向软骨细胞分化能力的影响。方法选择40只4周龄雌性SD大鼠,体质量约50 g。采用酶消化-密度梯度离心法提取脂肪干细胞,行体外培养,并通过流式细胞术鉴定脂肪干细胞。体外培养的脂肪干细胞分3组,即对照组、矿化组和GDF-5诱导组。不同试剂培养2周后分别进行免疫组织化学、甲苯胺蓝及免疫荧光染色。结果原代细胞经过接种沉降贴壁大量增殖后,按一定方向性排列呈旋涡状或束状,传代后细胞增殖迅速,生长稳定。细胞接种经Ⅱ型胶原免疫组织化学染色:GDF-5诱导组棕黄色阳性表达的细胞数量多;矿化组弱阳性,染色细胞数量少;对照组呈阴性表达。细胞甲苯胺蓝染色示:GDF-5诱导组蓝染阳性,细胞胞浆及其周围有紫红色异常染色;矿化组染色淡,染色细胞少;对照组阴性,细胞几乎均失染。结论GDF-5能够在体外增强脂肪干细胞的软骨化。

Objective To explore the effect of growth and differentiation factor-5(GDF-5)on chondrogenic differentiation of adipose-derived stem cells(ADSC).Methods Forty 4-week-old female SD rats with body mass of 50 g were sacrificed,the enzyme digestion density gradient centrifugation was used to extract ADSC and culture in vitro,and ADSC was identificated by flow cytometry.In vitro cultured ADSC were divided into 3 groups:control group,mineral group and GDF-5 induction group.Immunohistochemistry,toluidine blue and immunofluorescence staining were performed after 2-week cultured with different reagents.Results After inoculation,the primary cells were deposited and adhered to cell wall and mass proliferated,which lined in certain pattern of vortex or bundle.After passage,cells proliferated rapidly and grew stably.The immunocytochemistry results of typeⅡcollagen showed that there were large brown-yellow positive cells in GDF-5 induction group.In mineralization group,the staining cells were few.The control group was negative.Toluidine blue staining results showed that GDF-5 induction group blue staining was positive with abnormal purplish red staining of cytoplasm and surrounding cells.The mineralized group was light staining and few staining cells.The control group was negative and almost without staing.Conclusion It is demonstrated that GDF-5 can enhance chondrogenic differentiation of ADSC in vitro.