骨形态发生蛋白7诱导骨髓间充质细胞向软骨细胞分化的作用

目的 观察转染骨形态发生蛋白7(BMP7)基因的骨髓间充质细胞(BMSCs)所表达的BMP7蛋白在体外诱导该细胞向软骨细胞方向分化的作用.方法 分别取诱导液诱导的BMSCs(诱导组)、转染BMP7基因的BMSCs(转染组)以及未处理的BMSCs(对照组),在6孔板中细胞爬片,经HE、阿尔新蓝染色后,在倒置显微镜下观察形态.3组BMSCs培养7、14、21 d后,以半乳糖醛酸为对照品绘制标准曲线法测定培养液中糖胺多糖(GAG),ELISA法测定胶原蛋白Ⅱ.结果 HE、阿尔新蓝染色后,转染组和诱导组BMSCs分别呈红色和蓝色,具有软骨细胞特性.BMP7诱导后的细胞团表达了GAG和胶原蛋白Ⅱ.第21天,转染组GAG为(39.5±5.4)mg/L,胶原蛋白Ⅱ为(152.8±14.5)μg/L;诱导组GAG为(40.8±6.1)mg/L,胶原蛋白Ⅱ为(155.5±19.3)μg/L;对照组GAG为(17.1±3.4)mg/L,胶原蛋白Ⅱ为(89.7±14.3)μg/L;与对照组比较,转染组与诱导组GAG、胶原蛋白Ⅱ水平明显增高(P均<0.05),转染组与诱导组比较,则未见明显改变(P>0.05).结论 转染BMP7基因的BMSCs表达的活性BMP7能够诱导BMSCs向软骨细胞方向分化,且其诱导水平达到了诱导液的诱导水平.

Bone morphogenetic protein 7 induced bone marrow-derived mesenchymal cells differentiating into chondrocytes

Objective To examine the induction effects of bone marrow mesenchymal stem cells(BMSCs) transfected with bone morphogenetic protein 7 (BMP7) gene differentiating into chondrocytes. Methods We observed the phenotype of cells which were stained with alcian blue and HE climbing to the six pore plate with invert microscope. The glycosaminoglycan (GAG) value in culture medium was detected in control group,BMP7 transfect and culture medium induced groups after 7,14 and 21 days using standard curve method. Standard curve was described using galacturonic-acid as reference substance. The content of collagen Ⅱ was detected by ELISA method. Results HE and Alcian blue staining showed that BMP7 gene transfection group and the group induced by fluid possess the characteristics of chondrocyte. BMP7 induced BMSCs differentiation to chondrocyte which secrete specific protein called collagen Ⅱ and GAG. Content of GAG were (17.1±3.4),(39.5±5.4),(40.8±6.1)mg/L in control group,BMP7 gene transfected group and induced group,collagen Ⅱ were (89.7±14.3),(152.8±14.5),(155.5± 19.3)μg/L in these three groups separately. Comparing with control group,GAG and collagen Ⅱ of BMP7 gene transfected group and culture medium induced group increased obviously(all P < 0.05),but there was no significant difference between BMP7 gene transfeeted group and culture medium induced group (P > 0.05). Conclusion This active protein induces BMSCs differentiating into chondrocyte,in a level similar to that of inducing medium.