8-异戊烯基柑橘素促进体外培养成骨细胞成熟矿化的研究

Effects of 8-Prenylnaringenin on Osteoblasts Differentiate and Biomineralization in vitro

研究8-异戊烯基柑橘素对体外培养大鼠颅骨成骨细胞(rat skull osteoblasts,ROB)的分化成熟及生物矿化的影响.取新生大鼠颅骨多次酶消化法得到成骨细胞,培养于含10%FBS的MEM培养液中,3天后首次换液,待细胞铺满皿底传代培养.以碱性磷酸酶(alkaline phosphatase,ALP)为检测指标,96孔板梯度筛选作用最佳浓度,在最佳浓度作用并成骨性诱导培养的第3、6、9、12天测ALP活性、钙盐沉积量;第12天进行ALP和钙化结节组织化学染色及计数;成骨性诱导后不同时间点提取Total RNA,RT real-time PCR法检测成纤维细胞生长因子(bFGF)、胰岛素样生长因子-1(IGF-1)、成骨相关转录因子Osterix、Runx-2和骨形态发生蛋白-2(BMP-2)的基因表达情况;成骨性诱导的第4、8、12天裂解获得细胞总蛋白,蛋白质印迹法检测人Ⅰ型胶原蛋白(COL-Ⅰ)的蛋白质表达量.研究结果表明:1×10-6 mol/L能显著促进成骨细胞的成熟分化,表现为提高ROB的ALP活性、促进钙盐沉积、增加钙化结节数量;提高bFGF、IGF-1、Osterix、Runx-2和BMP-2 mRNA表达水平;促进COL-Ⅰ的合成.由此可知终浓度为1×10-6 mol/L 8-异戊烯基柑橘素能显著促进ROB的分化成熟及生物矿化,证明8-异戊烯基柑橘素能促进成骨细胞的分化成熟及生物矿化,作为促进骨修复和抗骨质疏松的有效成分具有较大的药用价值.

To investigate the effects of 8-prenylnaringenin on osteoblasts in vitro under the conditions of the ability to differentiate and biomineralization.Neonatal SD rat skull was segregated,bone cells were obtained by enzyme digestion and cultured in MEM containing 10% FBS.Three days later the culture medium was changed at the first time,serial subcultivation was proceeded until cells covered with 90% culture dish.The 8-prenylnaringenin final concentration is 1×10-6 mol/L.Under the induce condition,the Alkaline phosphatase activity,osteocalcin and calcium salt sediment yield were measured at the 3rd,6th,9th,12th day.At the 12th day,histochemistry dyeing was carried for calcified tubercle and ALP.Total RNA was isolated and the gene expression of bFGF,IGF-1,osterix,BMP-2 and Runx-2 were investgated by RT-real time PCR.8-Prenylnaringenin significantly advanced osteoblasts to differentiate and biomineralization.Raised the ALP activity and calcium salt sediment yield and osteocalcin and COL-Ⅰ,increased calcified tubercle amount were manifested.Besides,8-Prenylnaringenin also can enhance the mRNA level of bFGF,IGF-1,osterix,BMP-2 and Runx-2.The 8-prenylnaringenin with final concentration 1×10-6 mol/L can predominantly promote ROB differentiation and biomineralization.