骨桥蛋白在体外诱导大鼠骨髓基质干细胞向成骨细胞分化中的作用

Effects of osteopontin in mice bone marrow-derived mesenchymal stem cells differentiating into osteoblasts in vitro

目的:观察骨桥蛋白是否能诱导体外培养的大鼠骨髓基质干细胞(BMSCs)向成骨细胞分化,探讨其在骨折愈合过程中的作用机制,为其以后在骨折愈合方面的研究提供确切的理论依据。方法:以冲洗法获取Wistar大鼠骨髓,用贴壁法分离提纯BMSCs,传至第3代后分组,体外培养的BMSCs分为实验组和对照组,实验组添加含0.2nmol/L骨桥蛋白的DMEM条件培养基;对照组为不含骨桥蛋白的DMEM培养基,于不同时间点收集细胞。通过光镜观察培养的细胞形态,以细胞计数法测定生长曲线,应用流式细胞仪分析细胞周期,并行碱性磷酸酶(ALP)染色和活性测定及骨钙素(OCN)含量测定。结果:实验组细胞具有成骨细胞的形态学特征;实验组细胞生长曲线右移,细胞G0/G1期比例平均增加13.17%(P<0.01),S期比例减少74.93%(P<0.01);实验组ALP染色呈阳性,且ALP活性、OCN含量均较对照组明显提高。结论:骨桥蛋白对BMSCs有促进成骨分化的作用。

Objective：To explore whether osteopontin （OPN） can induce in vitro bone marrow-derived mesenchymal stem cells （BMSCs） to differentiate into osteoblasts,and explore its mechanism in the union of fracture to provide exact theoretical basis for its further study in the union of fracture. Methods：The bone marrow was separated from the bone of limbs of rats and isolated and then purified using adherence filtration. The passage cells of the third generation were divided into groups. BMSCs were cultured and divided into experimental group and control group. In the experimental group,the culture medi-um contained 0.2 nmol/L OPN. Cell morphology was observed by the light microscope,cell growth curve was tested by cell number counting method,cell cycle was examined with flow cytometry. Alkaline phosphatase （ALP） staining and activity,osteocalcin （OCN） content were measured. Results：The experimental group cells had osteoblasts-like morphology; cell growth curve of experimental group moved rightward,fluorescence at G0/G1 phase increased 13.17% （P0.01） and that at S phase decreased 74.93% （P0.01）; ALP staining of experimental group cells was positive,ALP activity and OCN content of experimental group cells increased obviously compared with that of control group. Conclusion：OPN can improve the osteogenic differentiation of BMSCs.