雌激素水平对大豆苷原(DAI)促成大鼠骨细胞分化作用的影响  

Influence of estrogen levels in medium on actions of daidzein(DAI)in osteoblast differentiation of rats in vitro

作　　者：丁巧灵[1] 王金凤[1] 徐小雅[1] 周轶[1] 金慰芳[1] 王洪复[1] 高建军[1] 

机构地区：[1]复旦大学放射医学研究所骨代谢研究室,上海200032

出　　处：《复旦学报（医学版）》2015年第2期191-197,203,共8页Fudan University Journal of Medical Sciences

摘　　要：目的 观察基础雌激素（estrogen）水平对大豆苷原（daizein,DAI）促成骨细胞分化作用的影响。方法采用酶消化法分离培养新生SD大鼠头盖骨成骨细胞,通过培养液中添加17β-雌二醇改变培养微环境（包括空白对照）雌激素水平,应用MTT法和对硝基苯磷酸盐（p-nitropheny-phosate,PNPP）法检测细胞增殖率和碱性磷酸酶（alkaline phosphatase,ALP）活性,应用real-time PCR法检测其雌激素受体α（ERa）、雌激素受体β（ERβ）和过氧化物酶体增殖物激活受体（peroxisome proliferator-activated receptor,PPAR）γ的mRNA水平变化,研究雌激素基础水平变化对DAI促成骨细胞分化作用的影响。结果 培养环境中雌二醇水平升高致DAI的促分化作用减弱。其中100nmol/L DAI组ALP比活性较对照组的增幅由雌激素补充前的23%分别降至8%（17β-雌二醇为100pmol/L）和-11%（17β-雌二醇为1 000pmol/L）,显示基础雌激素水平对DAI促分化的拮抗作用。为避免培养液中血清雌激素的可能影响,进一步采用去激素血清培养（含2%去激素胎牛血清）。此时补充17β-雌二醇至100pmol/L,DAI各剂量组细胞ALP比活性较对照组明显增加（16%~21%,P〈0.05）。继续补充雌激素至1 000和10 000pmol/L时,其作用反而减弱。该结果显示,DAI促成骨细胞分化作用与雌激素基础水平有关,低雌激素状态（≤100pmol/L）可增强其作用。为进一步探索其作用机制,我们初步观察了雌激素（100pmol/L）和DAI（100nmol/L）单独或联合作用下成骨细胞ERa、ERβ和PPARγ受体表达变化。结果显示,100pmol/L 17β-雌二醇明显上调ERβ表达,并部分抵抗DAI下调ERβ的作用。结论 DAI的促成骨分化作用与基础雌激素水平有关,低雌激素（≤100pmol/L）状态有利于DAI的促分化作用,而雌激素水平升高可减弱其促分化作用。Objective To investigate the contribution of circulating estrogen to the action of daidzein（DAI）on osteoblast differentiation invitro. Methods Osteoblasts were prepared from calvaria of neonatal SD rats by sequential collagenase digestion and treated with DAI in MEM medium supplied with 2%fetal bovine serum（FBS）or 2% coat-striped fetal bovine serum（CSFBS）respectively.17β-estradiol was added to all medium to modify the estrogen levels as required.The cell proliferation and activity of alkaline phosphatase（ALP） were measured by 3-（4,5-dimethythiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT）and p-nitropheny-phosate（PNPP）assays respectively.The expression of ERa、ERβand peroxisome proliferator-activated receptor（PPAR）γwere determined by real-time RT-PCR. Results Daidzein promoted osteoblasts differentiation in vitro,which was modified by estrogen levels in medium.Compared to the control,the cell ALP activity（D405/D570）was increased to 23%（P〈0.001）by 100nmol/L of daidzein in medium containing with 2% FBS.The increase was fell down to 8%in presence of 100pmol/L 17β-estradiol and-11%in presence of 1 000pmol/L.When cultured in MEM medium containing with 2% CSFBS,the cell ALP activities,which were slightly increased by 1-100nmol/L of daidzein,were obviously increased by 16%-21%（P〈0.05）in presence of 100pmol/L 17β-estradiol.The promotions was weaken by the continued increase of estrogen in medium.The expressions of ERαwere dramatically down regulated by 100nmol/L of daidzein alone or the combination with 100pmol/L of estradiol.The transcriptional levels of ERβwere markedly increased（up to 2.5times）by estradiol,but decreased to 18%（P〈0.05）by daidzein,while back to 67%（P〉0.05）by the combination.The expressions of PPARγwere not altered significantly in transcriptional levels. Conclusions The results indicated that effects of daidzein in osteogenesis was modified by the levels of estrogen in medium and the optimal lower level of estrogen（≤100pmol/L�

关 键 词：成骨细胞 大豆苷原(DAI) 雌激素 碱性磷酸酶(ALP) 大鼠 

分 类 号：R336[医药卫生—人体生理学] R589.5[医药卫生—基础医学]