CAJ | 학술논문

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。
목적：연구음양곽감대효원수응효삼유입체배양적신생대서로골성골세포（ ROB）분화화성숙적영향。방법：취신생SD 대서로골，매소화법배양ROB，장전대1차적ROB포매우2 mg／mL적Ⅰ형서미효원중진행삼유입체배양，48 h후진행쌍을산형광소／전화병정（ FDA／PI ）세포염색、소목소－이홍（ HE ）염색，병용소묘전경관찰세포생장상태。분별채용종농도위1×10－4、1×10－5、1×10－6화1×10－7 mol／L적음양곽감진행간예，배양3、6、9 d후분별검측세포내감성린산매활성，사선최가약물농도；이최가농도음양곽감간예삼유응효중적ROB 12、24、36、48 h후，용실시정량PCR화단백질인적법분별검측골형태발생단백2（ BMP－2）、Runt상관전록인자2（RUNX－2）、Osterix mRNA급기단백표체정황。결과：효원수응효삼유입체배양적ROB 채용FDA／PI염색、HE염색이급소묘전경결구현시， ROB재효원수응효중분포균균，생장상태량호。종농도위1×10－5、1×10－6、1×10－7 mol／L적음양곽감가현저제고삼유응효중ROB적감성린산매활성，기중이1×10－6 mol／L농도음양곽감간예후ROB 적감성린산매활성최고。 1×10－6 mol／L 음양곽감능현저제고BMP－2、RUNX－2、Osterix mRNA급기단백표체량。결론：음양곽감가현저촉진삼유입체배양적ROB성골성상관인자적표체，제시음양곽감대효원수응효삼유입체배양적ROB구유교강적촉골형성활성。
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 .