生长分化因子5诱导脂肪干细胞向成软骨细胞方向分化的实验研究

Experimental study of growth differentiation factor 5 induced adipose-derived stem cells chondrogenic phenotype in vitro

目的通过质粒转染探讨生长分化因子5(GDF-5)诱导大鼠脂肪干细胞向成软骨细胞方向分化的影响。方法选取雄性SD大鼠采用酶消化-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,即转染组、空质粒组和对照组。转染组采用Lipofectamine^(TM)2000进行脂质体pcDNA3.1(+)/GDF-5重组质粒瞬间转染;空质粒组采用空质粒pcDNA3.1(+);对照组只加入等量脂质体。转染后观察各组细胞形态。同时,通过免疫组织化学、免疫荧光检测培养2周后Ⅱ型胶原表达水平,通过甲苯胺蓝染色检测2周后蛋白聚糖表达水平。结果转染2周后,空质粒组、对照组细胞形态未见明显变化,转染组长梭形的细胞明显向成软骨的多角形方向变化。Ⅱ型胶原经免疫组织化学染色,转染组可见棕黄色染色,空质粒组、对照组无明显染色;免疫荧光结果显示,转染组细胞呈亮绿色,空质粒组、对照组均未见明显染性着色。蛋白聚糖经甲苯胺蓝染色后转染组细胞呈蓝染,空质粒组、对照组均未见明显染性着色。结论pcDNA3.1(+)/GDF-5转染脂肪干细胞能显著增加Ⅱ型胶原、蛋白聚糖的表达,促进脂肪干细胞向成软骨细胞方向分化。

Objective To explore the inductive effect of growth differentiation factor-5（GDF-5） on differentiation of rat adiposederived stem cells（ADSC） into chondrocytes by plasmid transfection. Methods Forty male SD rats with 4-week-old were selected, the enzyme digestion density gradient centrifugation was used to extract ADSC and culture in vitro, and identificated ADSC by flow cytometry. In vitro culture of ADSC were divided into 3 groups： transfection group, blank plasmid group and control group. The transfection group was used LipofectamineTM2000 transfected liposome pcDNA3.1（＋）/GDF-5 recombinant plasmid; blank plasmid group was transfected blank pcDNA3.1（＋）; control group was added equal volume of liposome. The morphology of each group was observed after transfection. The expression of type Ⅱ collagen was detected by immunohistochemistry and immunofluorescence after cultured 2-week, and the expression level of proteoglycans was measured by toluidine blue staining after 2-week. Results After transfected 2-week, the cell morphology in blank plasmid group and control group was no significant changed, the long spindle-shaped cells were significantly changed cartilage in transfection group. In immunocytochemistry results of type Ⅱ collagen, the transfection group occurred brown yellow staining, the blank plasmid group and control group was no obvious staining. The immunofluorescence results indicated transfection group occurred light green, the blank plasmid group and control group was no obvious staining. Toluidine blue staining results showed transfection group were stained blue, the blank plasmid group and control group was no metachromasia staining.Conclusion Transfection of pcDNA3.1（＋）/GDF-5 to ADSC could significantly increase expression of type Ⅱ collagen and proteoglycan, and promote the chondrogenic differentiation of ADSC.