大豆苷元对原代培养大鼠成骨细胞功能的影响

Effect of daidzein on osteoblasts function of rats cultured primarily in vitro

目的:了解大豆苷元对原代培养大鼠成骨细胞增殖、分化、钙含量及矿化功能的影响。方法:实验于2004-08/2005-06在北京同仁医院检验科完成。选取出生24h以内的SD大鼠30只,断颈处死,无菌取头盖骨,获取成骨细胞进行培养。分为对照组、大豆苷元10μmol/L组、大豆苷元5μmol/L组和大豆苷元1μmol/L组。应用四甲基偶氮唑盐法、对硝基苯磷酸盐法、原子吸收分光光度法及茜素红染色方法观察大豆苷元对体外培养成骨细胞的增殖、碱性磷酸酶表达、基质钙含量及矿化结节形成的影响。结果:①各浓度组与对照组相比均可增加吸光度值,刺激成骨细胞的增殖,其中大豆苷元10μmol/L组与对照组相比,差异有显著性意义(大豆苷元10μmol/L组为0.82130±0.10130,大豆苷元5μmol/L组为0.66370±0.04749,大豆苷元1μmol/L组为0.67750±0.05392,对照组为0.66250±0.07382,P<0.005)。②各浓度组大豆苷元作用于成骨细胞48h,均可使碱性磷酸酶活性增加,差异有显著性意义(大豆苷元10μmol/L组为1.10917±0.34190,大豆苷元5μmol/L组为0.97433±0.33267,大豆苷元1μmol/L组为0.84033±0.20408,对照组为0.55050±0.15489,P<0.005或0.02);各浓度组大豆苷元作用于成骨细胞72h,均可使碱性磷酸酶活性增加,差异有显著性意义(大豆苷元10μmol/L组为1.04667±0.12217,大豆苷元5μmol/L组为0.95000±0.07330,大豆苷元1μmol/L组为1.07700±0.14704,对照组为0.81867±0.07829,P<0.005或0.02)。③各浓度组大豆苷元处理细胞18d,可增加细胞基质钙含量,其中大豆苷元5μmol/L组与对照组相比,差异有显著性意义犤大豆苷元10μmol/L组(0.69975±0.13809)mg/L,大豆苷元5μmol/L组(1.01225±0.13277)mg/L,大豆苷元1μmol/L组(0.70375±0.07146)mg/L,对照组(0.60000±0.10494)mg/L,P<0.005犦。④大豆苷元1μmol/L和5μmol/L组处理细胞18d,形成矿化结节数高于对照组犤大豆苷元5μmol/L组(182.7±30.1)个,大豆苷元1μmol/L组(117.0±41.9)个,对照组(74.7±9.5)个,P<0.005犦。结论:大豆苷元具有刺激成骨细胞增殖,提高碱性磷酸酶活性、细胞基质钙含量及矿化结节形成的数量的作用。

AIM： To study the effects of daidzein on proliferation, differentiation, calcium content and mineralization of osteoblasts of rats cultured in vitro. METHODS：The experiment was conducted in the Department of Medical Laboratory, Rentong Hospital between August 2004 and June 2005. Totally 30 SD rats born in 24 hours were selected and killed by cutting the neck to culture the osteoblasts after taking the parietal bone sterilely. The cells were divided into control group, 10, 5 and 1 μmol/L daidzein groups. MTT, PNPP, atomic absorptiometry and alizarin red s （ARS） were used to observe the effect of daidzein on proliferation, activity of alkaline phosphatase （ALP）, contents of matrix calcium and number of mineral nodes of osteoblasts cultured in vitro. RESULTS： ①Compared with the control group, all doses of daidzein could increase absorbance value and proliferation of osteoblasts, and there were Significant differences between the control group and the 10 μmol/L daidzein group （10 μmol/L daidzein group： 0.821 30±0.101 30; 5 μmol/L daidzein group 0.663 70±0047 49; 1μmol/L daidzein group 0.677 5±0.053 92; the control group： 0.662 50±0.073 82, P 〈 0.005）. ②After treating for 48 hours and 72 hours, the ALP activity was increased by all doses of daidzein groups, which had significant differences （after 48 hours： 10 μmol/L daidzein group： 1.109 17±0.341 90; 5 μmol/L daidzein group： 0.974 33±0.332 67; 1 μmol/L daidzein group： 0.840 33±0.204 08; the control group： 0.550 50±0.154 89, P 〈 0.005 or 0.02; after 72 hours： 10 μmol/L daidzein group： 1.046 67±0.122 17; 5 μmol/L daidzein group： 0.950 00±0.073 30; 1 μmol/L daidzein group： 1.077 00±0.147 04; the control group： 0.818 67±0.078 29, P 〈 0.005 or 0.02 ）.③After cultured cells for 18 days, the content of matrix calcium was increased by all doses of daidzein, and there were significant differences between the 5 μmol/L daidzein group and the control group [10 μmol/L daidzein group： （0.699 75±0.138 09） mg/L; 5 μmnol/L daidzein group （1.012 25±0.132 77） mg/L; 1 μmol/L daidzein group： （0.703 75±0.071 46） mg/L; the control group： （0.600 00±0.104 94） mg/L, P 〈 0.005].④ After treating cells for 18 days, the number of mineral nodes of the 1 and 5 μmol/L daidzein groups was higher than the＇control group [5 μmol/L daidzein group： （182.7±30.1）; 1μmol/L daidzein group： （117.0± 1.9）; the control group： （74.7±9.5）, P〈 0.005]. CONCLUSION： Daidzein can stimulate the proliferation of osteoblasts, improve the ALP activity, and increase the content of matrix calcium and number of mineral nodes.