联合rhBMP-2与成骨细胞诱导骨髓基质细胞成骨分化的实验研究

目的探讨rhBMP-2和成骨细胞联合诱导骨髓基质细胞(BMSCs)的成骨分化能力。方法应用全骨髓贴壁法分离培养4周龄SD大鼠的BMSCs,通过改进酶消化法结合反复贴壁法培养1日龄SD乳鼠成骨细胞。实验分为4组:rhBMP-2诱导组、成骨细胞诱导组、rhBMP-2+成骨细胞诱导组、单纯培养液组。倒置相差显微镜及扫描电镜观察细胞生长情况;分别收集第3、6、9天细胞培养液上清液定性及定量测定碱性磷酸酶并进行统计学分析。结果 rhBMP-2诱导组、成骨细胞诱导组、rhBMP-2+成骨细胞诱导组均检测出成骨细胞生成,其中,rhBMP-2+成骨细胞诱导组碱性磷酸酶含量明显大于前两组,差异有统计学意义(P0.05)。结论rhBMP-2、成骨细胞、rhBMP-2联合成骨细胞均能提供成骨微环境,并在体外诱导BMSCs向成骨细胞分化,rhBMP-2联合成骨细胞对BMSCs的成骨分化具有更优的诱导能力。

Experiment research of combined rhBMP-2 and osteoblast inducing osteogenesis of bone mar-row stromal cell

Objective To investigate the osteogenesis abilities of the combined effects of rhBMP-2 and osteoblasts on BMSCs. Methods BMSCs from 4-week-old SD rat were isolated and cultured by whole bone marrow adherence method. The newly born （1d） SD rats were sacrificed and osteoblasts were obtained by modified enzymatic digestion and differential attachment technique. There were four experiment groups： rhBMP-2 induced group, osteoblasts in-duced group, rhBMP＋ osteoblasts induced group, and medium group. Morphology observations of cells were per-formed through inverted phase contrast microscope and scan electron microscope. The biological characteristic assays of alkaline phosphatase （ ALP） were performed. The cell culture supernatant was collected to detect the content of ALP in 3d,6d and 9d. Results Osteoblast-like cells were observed in rhBMP-2 induced group, osteoblasts induced group, rhBMP＋ osteoblasts induced group, and the content of of ALP in rhBMP＋ osteoblasts induced group was higher than that in rhBMP-2 induced group and osteoblasts induced group significantly （ P〈0. 05 ） . Conclusions Osteoblasts, rhBMP-2 can provide osteogenic microenvironment to induce the differentiation of BMSCs into osteoblasts in vitro. The rhBMP-2 combined with osteoblasts has better ability to induce the differentiation of BMSCs into osteo-blasts in vitro.