17β-雌二醇对大鼠骨髓间充质干细胞成骨分化的作用

Effect of 17β-estradiol on osteogenesis differention of mesenchymal stem cells in rats

目的:探讨不同剂量17β-雌二醇(17β-E2)对大鼠骨髓间充质干细胞(MSCs)成骨分化和骨钙素(BGP)基因表达的影响,为应用17β-E2调控MSCs向成骨细胞分化提供依据。方法:应用密度梯度离心法与贴壁筛选法分离出MSCs,连续传代3次,进行成骨细胞的诱导分化。MSCs分为对照组[单纯成骨细胞培养基(OBM)培养]和不同剂量17β-E2组(在成骨诱导分化液中分别添加0.1、1.0、10.0和100.0pmol·L-117β-E2)。各组细胞在第7天利用放射免疫法测定碱性磷酸酶(ALP)活性、Von Kossa染色检测钙盐沉积;第14天应用酶联免疫吸附法测定Ⅰ型胶原(ColⅠ)水平;第21天应用实时荧光定量PCR检测BGP mRNA表达水平,采用茜素红染色及在酶标仪上560nm波长下测定各组细胞的吸光度(A)值,定量分析成骨矿化能力。结果:应用密度梯度离心法与贴壁筛选法成功分离出MSCs,细胞呈成纤维细胞样,椭圆形核,核仁可见。传代培养的MSCs生长旺盛,保持原代细胞的形态特征。与对照组比较,不同剂量17β-E2组细胞ALP活性和ColⅠ水平增加(P<0.05或P<0.01),骨基质的钙化能力增强(P<0.05或P<0.01),并呈剂量依赖性;100.0pmol·L-1 17β-E2组BGP mRNA表达水平升高(P<0.01)。结论:密度梯度与贴壁筛选方法分离所得细胞为大鼠MSCs,17β-E2可增强MSCs成骨分化能力,上调BGP mRNA表达,并呈剂量依赖关系。

Objective To observe the influence of different doses of 17β-estradiol（17β-E2）on the osteogenesis differention of bone marrow mesenchymal stem cells（MSCs）and the expression of bone gamma-carboxyglutamicacid-containing proteins（BGP）gene,and to clarify the mechanism of estrogen replacement treatment for postmenopausal osteoporosis.Methods The MSCs were isolated by density gradient centrifugation and adherence screening method for continuous three passages,and the osteogenic induction and differention were performed.The MSCs were divided into control group（OBM）and different doses of 17β-E2groups（anding 0.1,1.0,10.0and100.0pmol·L^-1 17β-E2 in osteogenic induction liquid）.On the 7th day,radioimmunoassay was used to detect to alkaline phosphatase（ALP）activity,Von Kossa staining for calcium deposition of the cells in various groups.On the 14 th day,the collagenⅠ（ColⅠ）level was determined by enzyme linked immunosorbent assay.On the 21 th day,the BGP mRNA expression level was detected by real-time fluorescence quantitative PCR,and the cells＇ optical density（A）at 560 nm was determined.Results The MSCs were isolated successfully by density gradient centrifugation and adherence screening method.The MSCs appeared fibroblasts-like with ellipse nucleus with one to two nucleolus.The subculturing MSCs grew well and maintained the morphological characteristics of primary cells.Compared with control group,the ALP activities and Col Ⅰ levels in different doses of 17β-E2 groups were increased（P〈0.05 or P〈0.01）,and the bone matrix calcification was enhanced in a dose-dependent manner（P〈0.05 or P〈0.01）;the BGP mRNA expression level in 100.0pmol·L^-1 17β-E2 group was increased（P〈0.01）.There were significant differences in the BGPmRNA expression levels difference between 10.0 and100.0pmol·L^-1 groups（P〈0.05）ALP.The col-I levels and bone matrix calcification had significant differences between various groups（P〈0.05）.Conclusion The separated cells in this study are the rat MSCs.17β-E2 can enhance the osteogenesis differention of MSCs and up-regulate the BGP mRNA expression in a dose-dependent manner.