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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 investigate the effect of GDF-5 on the differentiation of rat adipose derived stem cells into chondrogenic cells by plasmid transfection. Methods Adipose-derived stem cells were isolated from male Sprague-Dawley rats by enzymatic digestion-density gradient centrifugation and cultured in vitro. Adipose-derived stem cells were identified by flow cytometry. Adipose-derived stem cells cultured in vitro were divided into 3 groups: transfection group, empty plasmid group and control group. The transfection group was transfected with pcDNA3.1 (+) / GDF-5 recombinant plasmid by lipofectamine TM 2000. The empty plasmid pcDNA3.1 (+ Plastids. After transfection, the morphology of each group was observed. At the same time, the expression of type Ⅱ collagen was detected by immunohistochemistry and immunofluorescence two weeks after the culture, and the expression of proteoglycan was detected by toluidine blue staining two weeks later. Results After 2 weeks of transfection, there was no significant change in the cell morphology of the empty plasmid group and the control group. The long spindle cells in the transfected group obviously changed to the polygonal shape of cartilage. Immunohistochemical staining showed that the type Ⅱ collagen was stained brown and yellow in the transfection group, but there was no obvious staining in the empty plasmid group and the control group. The immunofluorescence results showed that the cells in the transfection group showed a bright green color and the empty plasmid group and the control group showed no obvious staining Dyeing coloring. Proteoglycan staining by toluidine blue cells transfected cells were blue dye, empty plasmid group, the control group were no significant staining. CONCLUSION: Adipose-derived stem cells transfected with pcDNA3.1 (+) / GDF-5 can significantly increase the expression of type II collagen and proteoglycan and promote the differentiation of adipose-derived stem cells into chondrogenic cells.