成骨因子BMP7在骨膜细胞体外培养中的作用

目的 探讨成骨因子骨形态发生蛋白(BMP7)在骨膜细胞体外培养中的诱导分化作用.方法取材于成人胫骨骨膜,常规细胞培养法行骨膜细胞体外培养,分为实验组和对照组,分别加入BMP7加成骨细胞培养辅助剂和单纯成骨细胞培养辅助剂,相差显微镜观察骨膜细胞形态特征.每组分别在第5、10、15、20天设3个样本,采用ALP试剂盒法及钙结节Von Kossa染色法分别检测成骨细胞特异性标志物ALP和钙结节的表达情况.结果 骨膜细胞在体外生长良好,实验组和对照组形成的成骨细胞增殖良好,形态一致.细胞早期呈梭形,饱满透明,立体感强;分裂期呈立方形或短柱状;后期由长梭形逐渐变成宽梭和不规则形.由BMP7诱导的骨膜细胞的成骨标志物ALP和钙结节染色阳性率明显高于对照组,有统计学意义(P＜0.01).结论 骨膜细胞具有良好的成骨和再生能力,BMP7在体外培养中具有明显增强骨膜细胞分化为成骨细胞的作用.

The role of BMP7 in periosteal cells in vitro

Objective To study the role of bone morphogenetic protein-7 in the osteogenic differentiation of periosteal cellsin vitro. Methods Periosteal cells, obtained from adult tibial periosteum, were cultured by routine method in vitro, and divided into two groups. One group cultured with BMP7 and the supplements of 100 nmol dexametasone, 10 mmol b-glycerophosphate and 50 mg/mL L-ascorbic acid (BMP7 group), the other cultured with the supplements alone as the control (control group). Ultrastructure and morphological changes of periosteal cells were observed by contrast phase microscope and electron microscope. In order to test the expression of markers of osteoblastic differantiation in periosteal cells, involved mineralized node and alkaline phosphatase. Each group was tested at the time of 5 d, 10 d, 15 d, 20 d, respectively, using ALP kit stain and Von Kossa stain with 3 samples at each time. Results The periosteal cells cultured by routine method and induced into osteoblast differentiation with BMP7 were both growing well, in vitro. Microscope observations showed that the periosteal cells were spindle-shaped, well-stacked, transparent and three-dimensional in the early stage, and cube-shaped or puncheon shaped in the mitotic phase, gradually became wide shuttle and irregular shape with a lot secretion in telophase. The positive cells were visible by the ALP kit staining and Von Kossa staining of calcium nodules at 5 d, 10 d, 15 d and 20 d in both groups.A difference of positive rate at each time point was found between BMP7 group and control group at 5 d, 10 d, 15 d, 20 d, and the difference was statistically significant (P ＜ 0.01). Conclusion It displayed well regeneration and osteogenesis ability in the periosteal cell. BMP7 has definite osteo-inductive activity, which can obviously enhance the proliferation and ossifyng differentiation of periosteal cells.