脱水淫羊藿素与山柰素对体外培养成骨细胞成熟矿化影响的比较研究

比较研究脱水淫羊藿素与山柰素对体外培养大鼠成骨细胞(rat osteoblasts,ROB)增殖与成熟矿化的影响。取新生SD大鼠颅骨,多次酶消化法获得成骨细胞,培养于含10%FBS的MEM培养液中,3天后首次换液,铺满皿底90%以上后传代培养。分别用1×10 4、1×10 5、1×10 6和1×10 7mol.L 1的脱水淫羊藿素和山柰素进行药物干预,增殖分析采用MTT法,诱导培养9天后检测碱性磷酸酶(alkaline phosphatase,ALP)活性,筛选出最佳浓度。采用最佳浓度的脱水淫羊藿素和山柰素进行药物干预,比较脱水淫羊藿素、山柰素和对照组之间的ALP、钙盐沉积量、骨钙素分泌量、CFU-FALP和矿化结节数量等成骨性指标。提取总RNA,实时荧光定量PCR(real time PCR)检测BMP-2、Osterix(OSX)及Runx-2的mRNA表达情况,同时提取总蛋白,Western blotting法检测Ⅰ型胶原蛋白的分泌量。结果表明,1×10 4mol.L 1脱水淫羊藿素和山柰素对细胞增殖有抑制作用;1×10 5mol.L 1脱水淫羊藿素虽对ROB增殖无明显影响,但能显著提高成骨细胞中ALP活性、钙盐沉积量和骨钙素分泌量,增加CFU-FALP和矿化结节数量,与成骨细胞成熟矿化相关的因子BMP-2、OSX和Runx-2的基因表达量也显著升高,同时I型胶原蛋白的分泌量也明显增多;而山柰素并没有表现出促进体外培养ROB成熟矿化的特性。脱水淫羊藿素促进成骨细胞成熟矿化的作用明显高于山柰素,很可能是由脱水淫羊藿素8位C原子上存在的异戊烯基引起的。

Comparison of effects of kaempferide and anhydroicaritin on biomineralization of cultured osteoblasts

This study is to compare the effects of kaempferide and anhydroicaritin on biomineralization of rat osteoblasts(ROB) in vitro.Calvarias were dissected aseptically from newborn SD rats,the osteoblasts were obtained by enzyme digestion and were cultured in MEM containing 10% FBS.The medium was changed every three days,and serial subculture was performed when cells covered with 90% of the dish.Kaempferide and anhydroicaritin were separately added with final concentrations of 1×10 4,1×10 5,1×10 6 and 1×10 7 mol.L 1 under the conditions of osteogenic differentiation.The proliferation was measured by MTT,and the optimal concentration was detected by the ALP activity at the 9th day after osteogenic induction culture.The osteogenic indexes of kaempferide,anhydroicaritin and control group with the optimal concentration were compared.The result showed that the anhydroicaritin at concentration of 1×10 5 mol.L 1 had significantly promoted the activity of ALP,calcium content and osteocalcin content,increased the number of CFU-FALP and mineralized nodules,enhanced the mRNA level of BMP-2,OSX and Runx-2,which are key genes of osteogenic differentiation,and raised the protein content of collagen-I.However,the kaempferide group had not significantly represented the ability that promoted osteogenic differentiation of ROB.The difference of osteogenic differentiation on ROB between kaempferide and anhydroicaritin was caused by the prenyl group on C-8 of icariin.