染料木黄酮对乳鼠颅盖骨成骨细胞增殖分化和转化生长因子β表达的影响  

Influences of genistein on the proliferation and differentiation of calvaria osteoblasts as well as transforming growth factor beta levels in neonatal rats

作　　者：张月红[1] 金宏[2] 许志勤[2] 南文考[2] 王先远[2] 薛长勇 高兰兴[2] 

机构地区：[1]解放军总医院营养科,北京市100853 [2]解放军军事医学科学院卫生学环境医学研究所,天津市300050

出　　处：《中国组织工程研究与临床康复》2007年第49期9937-9940,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:动物实验、临床研究以及流行病学调查表明染料木黄酮可预防骨质疏松的发生,但其具体机制尚不清楚。观察染料木黄酮对成骨细胞活性的影响及其与转化生长因子β的关系。方法:实验于2001-05/2003-05在卫生学环境医学研究所生化实验室完成。①实验材料:选择出生3d的Wistar大鼠,体质量(10±2)g。②实验方法:无菌条件下分离颅盖骨,剪碎,加入胰蛋白酶和Ⅱ型胶原酶,用含体积分数为0.1胎牛血清的F-12培养基重悬,调整细胞浓度后接种于25mL培养瓶中,Ⅱ代细胞用于实验。实验分为染料木黄酮组、雌激素组和对照组(吐温20),染料木黄酮组浓度分别为0.1,1,10μmol/L,雌激素组的浓度分别为0.1,1nmol/L。③实验评估:培养48,72h应用四甲基偶氮唑盐测定成骨细胞增殖;培养48h3H-胸腺嘧啶掺入实验测定DNA合成;免疫组织化学方法观察转化生长因子β的表达。结果:①染料木黄酮对成骨胞增殖的影响:与对照组相比,培养48h后0.1,1,10μmol/L染料木黄酮四甲基偶氮唑盐的吸光度值分别增加1.43,1.36,1.05倍;0.1,1nmol/L雌激素组分别增加1.00倍和0.84倍;培养72h,染料木黄酮3个浓度组分别增加1.46,1.13倍和0.93倍,雌激素组分别增加2.26倍和2.30倍;各组与对照组相比差异均有显著性(P<0.05)。②3H-胸腺嘧啶掺入实验:0.1,1,10μmol/L染料木黄酮3H-胸腺嘧啶掺入量分别比对照组增加6.45,11.29,0.47倍;0.1,1nmol/L雌激素组增加16.5倍和15.4倍,各组与对照组相比差异均有显著性(P<0.05)。③成骨细胞转化生长因子β的表达:各组成骨细胞周围均有较强的呈棕色的阳性颗粒,但染料木黄酮组与对照组相比差异无显著性(P>0.05)。结论:不同浓度染料木黄酮和雌激素一样可以促进成骨细胞增殖和DNA合成,但染料木黄酮不是通过影响转化生长因子β的表达来促进成骨细胞增殖与分化,详细机制还需进一步研究。AIM： Animal and human study and epidemic data show that genistein prevent the onset of osteoporosis, but the involved mechanism is unclear. We studied the effects of genistein on osteoblasts activity and the role of the transforming growth factor-b in this process. METHODS： Experiments were performed at the Biochemical Laboratory in the Institute of Health and Environment Medicine from May 2001 to May 2003. （1）Wistar rats aged 3 days weighting （10±2）g were selected. （2）Calvarias were aseptically isolated and cut into pieces, and then digested through trypsin and collagenase Ⅱ. Prepitation was suspended with F-12 culture medium containing 0.1 volume fraction of fatal bovine serum and then cultured in 25 mL culture bottle after adjusting cell density. The second generation of cells was used in the experiment. The experiment was divided into a control （tween 20） group, a genistein group and an estrogen group. Doses of genistein were 0.1,1, 10 μmol/L, and the concentration of estrogen was 0.1,1 nmol/L, respectively. ③The proliferation of osteoblasts was assayed by MTT method at hours 48 and 72, and ^3H-TdR incorporation was used to synthesize DNA at hour 48. Transforming growth factor-β was measured by immunohistochemical assay. RESULTS： （1）Compared with the control group, the absorbance of MTT in the genistein of 0.1,1,10 μmol/L group was significantly increased 1.43,1.36,1.05 folds, and 1.46,1.13, 0.93 folds respectively after culturing 48 and 72 hours. The estrogen of 0.1,1 nmol/L groups increased 1.00, 0.84 folds and 2.26, 2.30 folds after culturing 48 and 72 hours, respectively （P 〈 0.05）. （2）The quantity of ^3H-TdR incorporation in the genistein of 0.1,1,10μmol/L groups significantly increased 6.45,11.29,0.47 folds respectively, while the estrogen 0.1,1 nmol/L groups increased 16.5 and 15.4 folds compared with control group （P 〈 0.05）. （3）There were a lot of positive brown particles around osteoblasts of all groups, but no significant difference was found b

关 键 词：染料木黄酮 成骨细胞 转化生长因子Β 

分 类 号：R681.4[医药卫生—骨科学]