生长分化因子5真核表达质粒转染小鼠骨髓基质干细胞体外诱导向软骨细胞分化

Mouse bone marrow stroma stem cells transfected by growth differentiation factor 5 eukaryotic expression plasmid induces chondrogenic differentiation in vitro

背景:生长分化因子5是软骨与骨组织形成、发育的重要调解因子,在诱导软骨形成,促进骨、软骨、肌健韧带损伤修复方面发挥重要作用。目的:小鼠骨髓基质干细胞体外转染真核表达质粒pcDNA3.1(+)/生长分化因子5,检测与软骨形成分化相关的细胞外基质及蛋白多糖的表达。设计、时间及地点:细胞学体外观察,于2008-03/12在武汉协和医院中心实验室完成。材料:雄性昆明种小鼠20只,由华中科技大学同济医学院实验动物中心提供。生长分化因子5真核表达质粒pcDNA3.1(+)/生长分化因子5为自备。方法:全骨髓贴壁法体外分离培养小鼠骨髓基质干细胞,取传至第3代细胞接种到6孔板,在细胞生长到90%融合时开始转染。设立3组:转染组采用LipofectamineTM2000进行脂质体介导pcDNA3.1(+)/生长分化因子5重组质粒瞬时转染;空质粒组转染空质粒pcDNA3.1(+);空白对照组只加入等量脂质体,其余步骤相同。主要观察指标:转染后72h通过RT-PCR及免疫细胞化学检测生长分化因子5基因与蛋白的表达鉴定转染是否成功,同法检测软骨基质Ⅱ型胶原的表达,转染后14d阿尔辛蓝染色检测蛋白聚糖的表达。结果:转染组有一大小为219bp的特异性扩增条带,骨髓基质干细胞胞浆内呈棕色阳性染色;而空质粒组、空白对照组均未发生生长分化因子5基因转录,无特异性扩增条带,且细胞胞浆未见明显染色。转染组可检测到Ⅱ型胶原基因的表达,基因大小225bp,Ⅱ型胶原细胞胞浆中可见棕黄色染色;空质粒组、空白对照组均未检测到Ⅱ型胶原基因的表达,SP染色均无明显染色。阿尔辛蓝染色后转染组细胞呈蓝染,空质粒组、空白对照组均未见明显异染性着色。结论:pcDNA3.1(+)/生长分化因子5转染骨髓基质干细胞能显著增加Ⅱ型胶原及蛋白聚糖的表达,促进骨髓基质干细胞向软骨细胞方向分化。

BACKGROUND： Growth differentiation factor 5 （GDF-5） is an important factor to regulate the formation and development of the cartilage and bone, it plays a crucial role on the promotion of repairing bone, cartilage and tendon ligament injury. OBJECTIVE： To transfect eukaryotic expression plasmid pcDNA 3.1 （＋）/GDF-5 to bone marrow stroma stem cells of mouse and to check the expression of extracellular matrix and proteoglycan which relates with the cartilage formation and differentiation. DESIGN, TIME AND SETTING： An in vitro observation regarding cells was performed in the central laboratory of Wuhan Union Hospital between March and December in 2008. MATERIALS： Twenty Kunming specimen male mice were offered by Experimental Animal Center of Tongji Medical College of Huazhong University of Science and Technology. The eukaryotic expression plasmid pcDNA 3.1（＋）/GDF-5 was preserved at the laboratory. METHODS： The marrow stroma stem cells were isolated from mouse bone marrow and cultured in vitro with whole bone marrow adherence method. Passage 3 cells were incubated on 6-well plate and began to transfect when they were 90% confluent. Experiment was assigned into three groups： transfection group underwent transient transfection of liposome-mediated pcDNA 3.1（＋）/GDF-5 using LipofectamineTi2000; blank plasmid group was transfeeted with blank plasmid pcDNA 3.1（＋）; control group was added with equal volume of liposome and other protocols were the same as above. MAIN OUTCOME MEASURES： The transfection efficacy was identified success by the expression of GDF-5 gene and protein using RT-PCR and immunocytochemistry at 72 hours following transfection, cartilage matrix Ⅱ collagen expression was determined as above methods. Then marrow stroma stern cells were cultured for additional two weeks to check expression of proteoglycan with alcian blue staining. RESULTS： In the transfection group, a 219-bp specific amplification band was visible, there were brown positive stain in the cytoplasm of marrow stroma stem cells; In blank plasmid group and control group, no GDF-5 transfection, specific amplification band or obvious stain of cytoplasm was observed. In the transfection group, the collagen Ⅱ gone was detected to express at 225 bp, with brown yellow stain in cytoplasm; in the blank plasmid group and control group, no collagen Ⅱ gone expression or SP .stain was observed. Alcian blue staining results showed the transfected cells were stained blue while those in the blank plasmid group and control group were not metachromasia stained. CONCLUSION： Gone transfection of pcDNA 3.1（＋）/GDF-5 to marrow stroma stem cells can significantly raise expression of collagen II and proteoglycan, and promote the chondrogenic differentiation of marrow stroma stern cells.