淫羊藿苷促进胶原水凝胶三维立体培养成骨细胞的成熟分化

目的:研究淫羊藿苷对胶原水凝胶三维立体培养的新生大鼠颅骨成骨细胞(ROB)分化和成熟的影响。方法:取新生SD 大鼠颅骨,酶消化法培养ROB,将传代1次的ROB包埋于2 mg/mL的Ⅰ型鼠尾胶原中进行三维立体培养,48 h后进行双乙酸荧光素/碘化丙啶(FDA/PI)细胞染色、苏木素-伊红(HE)染色,并用扫描电镜观察细胞生长状态。分别采用终浓度为1×10-4、1×10-5、1×10-6和1×10-7 mol/L的淫羊藿苷进行干预,培养3、6、9 d后分别检测细胞内碱性磷酸酶活性,筛选最佳药物浓度;以最佳浓度淫羊藿苷干预三维凝胶中的ROB 12、24、36、48 h后,用实时定量PCR和蛋白质印迹法分别检测骨形态发生蛋白2(BMP-2)、Runt相关转录因子2(RUNX-2)、Osterix mRNA及其蛋白表达情况。结果:胶原水凝胶三维立体培养的ROB 采用FDA/PI染色、HE染色以及扫描电镜结构显示,ROB在胶原水凝胶中分布均匀,生长状态良好。终浓度为1×10-5、1×10-6、1×10-7 mol/L的淫羊藿苷可显著提高三维凝胶中ROB的碱性磷酸酶活性,其中以1×10-6 mol/L 浓度淫羊藿苷干预后ROB的碱性磷酸酶活性最高。1×10-6 mol/L 淫羊藿苷能显著提高BMP-2、RUNX-2、Osterix mRNA及其蛋白表达量。结论:淫羊藿苷可显著促进三维立体培养的ROB成骨性相关因子的表达,提示淫羊藿苷对胶原水凝胶三维立体培养的ROB具有较强的促骨形成活性。
Abstract: Objective: To investigate the effects of icariin on the differentiation and maturation of rat calvarial osteoblasts(ROB) in collagen hydrogel three-dimensional culture. Methods: ROB were obtained by enzyme digestion from the segregated neonatal SD rats skull and were embedded in 2 mg/mL rat tail collagen for three-dimensional culture. The growth state of ROB was observed by FDA/PI staining, HE staining and scanning electron microscopy. ROB were treated with icariin at the concentration of 1×10-4, 1×10-5, 1×10-6 and 1×10-7 mol/L respectively. The activity of alkaline phosphatase(ALP) was detected after 3, 6, 9 d of icariin treatment. Three-dimensional cultured ROB were treated with optimal concentration icariin for 12, 24, 36, 48 h and total RNA was extracted and the mRNA expressions of bone morphogenetic protein-2(BMP-2), Runt-related transcription factor 2(RUNX-2) and Osterix were detected by real time RT-PCR. The protein expression of BMP-2, RUNX-2 and Osterix were examined by Western-blotting. Results: ROB were cultured in collagen hydrogel successfully. FDA/PI staining, HE staining, and scanning electron microscopy showed that ROB adhered with collagen tightly and distributed homogeneously. Icariin at final concentration of 1×10-5, 1×10-6 and 1×10-7 mol/L all enhanced the activity of ALP of collagen hydrogel three-dimensional cultured ROB, and 1×10-6 mol/L was the optimal concentration. Besides,icariin(1×10-6 mol/L) increased mRNA and protein expression of BMP-2、RUNX-2 and Osterix compared to control group. Conclusion: Icariin can enhance the expression of osteogenic markers of ROB in collagen hydrogel three-dimensional culture significantly. Key words: Extracellular matrix Epimedium brevicornum/administration & dosage Icariin Hydrogel Collagen Osteoblasts/drug effects Cells, cultured