补骨脂素与异补骨脂素对乳鼠颅骨成骨细胞分化成熟影响的比较研究

Comparative studies on the differentiation and maturation of rat calvarial osteoblasts by psoralen and isopsoralen in vitro

目的比较研究中药补骨脂中两种重要组分——补骨脂素与异补骨脂素对体外培养乳鼠颅骨成骨细胞(rat cal-varial osteoblasts,ROB)分化成熟的影响。方法取新生SD大鼠颅骨,多次酶消化法培养ROB,每3天换液1次,铺满80%皿底后传代培养。以1×10-5mol.L-1的补骨脂素和异补骨脂素对ROB的分化成熟进行药物干预,比较补骨脂素组、异补骨脂素组和不加药的对照组之间碱性磷酸酶(ALP)活性、碱性磷酸酶阳性克隆数(CFU-FALP)、骨钙素分泌量、钙盐沉积量以及钙化结节数量的差异,Real Time RT-PCR检测IGF-1、Osterix、Runx-2和collagen I的基因表达情况。West-ern blot法检测Ⅰ型胶原蛋白的分泌量。结果补骨脂素和异补骨脂素均可促进ROB的分化成熟,具体表现在明显提高ROB的ALP活性,促进钙盐的沉积以及骨钙素的分泌,增加CFU-FALP和钙化结节数量,提高IGF-1、Osterix、Runx-2和collagen I的mRNA水平,增强Ⅰ型胶原的蛋白表达,但异补骨脂素的活性明显高于补骨脂素。结论异补骨脂素促进ROB分化成熟的活性明显高于补骨脂素,提示异补骨脂素是中药补骨脂发挥抗骨质疏松活性的主要有效成分,具有开发为抗骨质疏松新药的潜力。

Aim To investigate the effects of psoralen and isopsoralen on the differentiation and maturation of rat calvarial osteoblasts（ROB）.Methods Neonatal SD rat skull was segregated to obtain ROB with enzyme digestion.The culture medium was replaced every three days.Serial subcultivation proceeded when cells were covered with 80% culture dish.Psoralen and isopsoralen were supplemented into the culture at 1×10-5 mol·L-1 respectively.The osteogenic differentiation markers including alkaline phosphatase（ALP） activity,CFU-FALP,osteocalcin secretion,calcium deposition and mineralized bone modulus were compared among the psoralen-supplemented group,the isopsoralen group and the control group.The gene expression of IGF-1,Osterix,Runx-2 and collagen I was examined by Real Time RT-PCR.Total protein was isolated and the secretion of collagenⅠwas examined by Western blot.Results Both psoralen and isopsoralen significantly improved ALP activity,CFU-FALP amount,osteocalcin secretion,calcium deposition and mineralized modulus.Besides,they enhanced the mRNA level of IGF-1,Os-terix,Runx-2 and collagen I,the expression of collagen Ⅰ was also promoted apparently.Isopsoralen was obviously stronger than pspralen in the above activities.Conclusion Isopsoralen is stronger than psoralen at enhancing the osteogenic differentiation of ROB,suggesting that isopsoralen is the main active component of Psoralea corylifolia L.and can be developed into a new drug for osteoporosis therapy.