8-异戊烯基柑橘素促进体外骨髓基质干细胞成骨性分化的研究

Investigation of the effect of 8-Prenylnaringenin on bonemarrow stromal cell's differentiation in vitro

目的研究8-异戊烯基柑橘素对体外培养大鼠骨髓基质干细胞(bone marrow stromal cells,BMSCs)的增殖与成骨性分化的影响。方法取大鼠股骨及胫骨全骨髓,利用全骨髓培养法培养于含体积分数为10%FBS的DMEM-F12培养液中。以碱性磷酸酶(alkaline phosphatase,ALP)为指标,96孔板梯度筛选作用最佳浓度。增殖分析采用MTT法。以1×10-6 mol.L-1的最佳浓度作用并成骨性诱导(1×10-7mmol.L-1地塞米松、10 mmol.L-1β-甘油磷酸钠和50 mg.L-1抗坏血酸)培养的d 4、8、12、16测ALP活性、钙盐沉积量,d 16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bFGF、IGF-1、Osterix、Runx-2、BMP-2及COL-Ⅰ的基因表达情况。结果 8-异戊烯基柑橘素不能促进BMSCs增殖,但8-异戊烯基柑橘素在1×10-6 mol.L-1时能促进其向成骨性分化,表现为提高BMSCs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、BMP-2及COL-Ⅰ的mRNA表达水平。结论 8-异戊烯基柑橘素可诱导骨髓间充质干细胞向成骨细胞分化,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。

Aim To investigate the effect of 8-Prenylnaringenin on bone marrow stromal cells in vitro under the conditions of the ability to differentiate into osteoblasts.Methods The rat bone marrow sample was obtained,and the all bone marrow cell culture methods were used to separate and collect the stromal cells.The cells were cultured in DMEM-F12 containing 10% fetal bovine serum.The 8-Prenylnaringenin final concentration was 1×10-6 mol·L-1.Under the induced condition,the Alkaline phosphatase activity,osteocalcin and calcium salt sediment yield were measured on the 4th,8th,12th,16th day.On the 16th day,histochemistry dyeing was proceeded for calcified tubercle and ALP.Total RNA was isolated and the gene expressions of bFGF,IGF-1,Osterix,BMP-2,COL-Ⅰand Runx-2 were investgated by RT-Real Time PCR.Results The bone marrow stromal cells were advanced to osteogenesis by 8-Prenylnaringenin,which was manifested by the raised ALP activity and calcium salt sediment yield and osteocalcin and increased calcified tubercle amount.Besides,it also could enhance the mRNA level of bFGF,IGF-1,Osterix,BMP-2,COL-Ⅰ and Runx-2.Conclusion The 8-Prenylnaringenin with the final concentration of 1×10-6 mol·L-1 can promote bone marrow stromal cell＇s differentiation to osteogenesis,which indicates its medicinal value in improving bone repair and anti-osteoporosis.