【作者】 刘毅； 张承昊； 吴术红；

【机构】 遵义医学院附属医院关节外科；

【摘要】 <正>目的:通过LFs对BMSCs进行诱导后细胞活性及增殖能力、韧带相关蛋白的分泌、细胞诱导效果。方法:通过密度梯度离心法获得大鼠BMSCs,通过组织块消化法获得LFs细胞,将细胞传至第三代。根据细胞的培养方式不同分为三组即A组骨髓间充质干细胞组,B组生长因子诱导组(bFGF-1和TGF-β,浓度均为10mg/ml),C组LFs诱导组,每组细胞均为1×105/孔。将三组细胞培养12天,并通过倒置显微镜观察细胞生长,MTT法绘制细胞生长曲线。在三组细胞培养第12天时,通过免疫组化法分析Ⅰ、Ⅲ型胶原蛋白的产量,通过天狼猩红和固绿染色观察细胞总胶原蛋白和非总胶原蛋白产量,通过实时荧光定量PCR检测韧带特异性基因的表达情况(Ⅰ、Ⅲ型胶原蛋白、韧粘素C、波形蛋更多还原

【Abstract】 Objective:To research that the changing of cellular activities,proliferation and EMC and remodeling related to ligament,effect of growth factors after LFs induced and assisted BMSCs.Methods:BMSCs were isolated and separated by Pereoll using intensity gradient centrifugation and selected.LFs were isolated and separated by collagenase.The cells,after they were passaged into P3,were divided into three groups,the BMSCs group,the growth factors induced group(bFGF-1 and TGF- β,10mg/ml) and LFs induced group,1 × 105/cell of 6-cell cultures.Cells activities and proliferation were observed by microscope and MTT.After 12 days culturing,The collagen type Ⅰ and type Ⅲ were determined by immunohistochemistry.Quantification of collagen and total extracellular matrix proteins were analyzed by selective binding of Sirius Red and Fast Green FCF.The mRNA expressions of all groups were analyzed by real-time RT-PCR(collagen type Ⅰ,Ⅲ,Fibronectin,Tenasein-C and MPP-2).Results:At the third passage,BMSCs were positive for CD29、 CD73、 CD90、 CD105,but negative for CD14、 CD19、 CD34、 CD45.To compare three groups,the LFS induced group had much higher activities and proliferation ability,they have more increasing in cellular proliferation,its 8.0%higher than the growth factors induced group and 17%higher than BMSCs group.The growth factors and LFs can induce BMSCs to enhance the concentration of the collagen type Ⅰ and type Ⅲ.The LFs induced group increased 11.4%and 8.3%respectively.The growth factor induced group increased 10.5%and 3.4%.The mRNA expression related to ligament of the induced groups(Collagen type Ⅰ、 Collagen type Ⅲ、Fibronection、MMP-2) increased.The mRNA of Collagen type I and Collagen type Ⅲ were similar,but the mRNA of Fibronection and MMP-2 of the LFS induced group was 1.3 times and 1.5 times than that of the growth factor induced group.Conclusion:LFs could induced BMSCs and its effect similar to the growth factor,and it was better in improving cells activities and enhancing mRNA relative with ligment.更多还原