低频电磁场干预大鼠骨髓间充质干细胞体外成软骨分化的研究

Chondrogenic potential of rat mesenchymal stem cells in vitro under the influence of electromagnetic fields

目的 探讨在体外条件下低频电磁场诱导大鼠骨髓间充质干细胞向软骨细胞分化的可行性。方法 大鼠骨髓间充质干细胞传代至第5代后,采用微团培养法在含成纤维生长因子-2和转化生长因子-β的培养基中生长,并给予正弦波电磁场（1.0 mT,50 Hz）干预。3周后进行阿利新蓝染色以检测软骨基质生成量,采用荧光定量聚合酶链反应（FQ-PCR）技术检测软骨特异性蛋白的基因表达水平,并使用二甲基-亚甲蓝（DMMB）染料结合法评估细胞微团中糖胺多糖水平。结果在电磁场和生长因子的协同作用下,大鼠骨髓间充质干细胞（BMSCs）细胞微团向软骨分化,细胞微团中Ⅱ型、X型胶原及蛋白多糖基因表达水平显著增加,而性别决定区Y框蛋白9（SOX9）的表达没有明显变化。与非暴磁组比较,暴磁组细胞糖胺多糖（GAG）/DNA比例较高（3.108±0.341）。结论电磁场促进大鼠BMSCs成软骨分化可能与细胞表达Ⅱ、X型胶原及糖胺多糖增多有关。电磁场在生长因子的协同作用下可诱导及维持间充质干细胞成软骨分化,但单因素电磁场刺激不能诱导大鼠骨髓间充质干细胞成软骨分化。

Objective To determine the chondrogenic potential of mesenchymal stem cells （BMSCs） under exposure of extremely low frequency electromagnetic fields （ELF-EMF） and discuss the application prospect of EMF in cartilage tissue engineering. Methods Cell pellets of rat bone marrow-derived BMSCs were cultured in vitro under the addition of fibroblast growth factor 2 （ FGF-2 ） and transforming growth factor-β3 （TGF-β3）. Pellet cultures were exposed to sinusoidal ELF-EMF （1.0 mT, 50 Hz ）. After 3 weeks of inductive differentiation culture, chondrogenesis was detected by alcian blue staining, and quantitative real-time polymerase chain reaction （real-time PCR） was used for detection of chondrogenesis related proteins. Glycosaminoglyean （GAG） contents of pellet cultures were determined using the dimethylmethylene blue （DMMB） dye-binding assay. Results With the effects of growth factors, EMF could significandy promote chondrogenic differentiation of BMSCs pellet cultures, and the gene expression of collagen type II, X and aggrecan in rat BMSCs was also significantly up-regulated. The gene expression of sex determining region Y box 2 （ SOX9 ） did not show significant difference between EMF exposed groups and nonexposed groups. The glycosaminoglyean （GAG）/DNA ratio of EMF exposed pellet cultures reached 3. 108 ± 0. 341 ,which was significantly higer than those unexposed pellet cultures. Conclusion Increased expression of collagen type II, X and GAG content in pellet cultures under EMF exposure may contribute to the chonderogenic differentiation of rat BMSCs. EMF was able to stimulate and maintain chondrogenic differentiation of rat BMSCs under influence of growth factors. EMF alone, however, could not induce chondrogenic differentiation.