目的：探讨1α，25-二羟维生素D3（1α，25-（OH）2D3）对体外培育SD大鼠成骨细胞（OB）增殖、分化和成熟矿化的影响。方法采用胰蛋白酶和胶原酶消化法从24h新生SD乳鼠颅盖骨分离得到的OB，设置0nmol/L、1nmol/L、10nmol/L、100nmol/L的1α，25-（OH）2D3为空白对照、低、中、高干预组，干预24h、48h和72h后分别采用噻唑蓝（MTT）比色法检测OB增殖率、PNPP法测定碱性磷酸酶（ALP）活性、放射免疫法检测骨钙素（OC）含量、RT-PCR检测BMP-2 mRNA表达；3d和6d后测定钙盐沉积量；10d和20d后茜素红染色法检测细胞钙化结节数。结果与空白组比较，在干预24h、48h、72h后低干预组成骨细胞A值分别增加20.50%、38.77%、18.22%，高干预组ALP分别增加（3.068±1.137）%、（0.505±0.156）%、（1.115±0.382）%，OC含量分别增加（0.285±0.097）%、（0.469±0.134）%、（0.734±0.225）%；高干预组成骨细胞钙沉积量在干预3d和6d后分别增加（0.968±0.236）%和（0.929±0.307）%，成骨细胞矿化结节在干预10 d和20d后，分别增加（13.889±3.973）%和（10.472±0.263）%，均呈正相关（P<0.05）。结论在本实验浓度范围内，中、高剂量1α，25-（OH）2D3可促进体外成骨细胞分化和矿化，刺激成骨细胞分泌骨钙素，发挥着促进骨形成的作用。%Objective To study the impaction of 1α,25-(OH)2D3 on the proliferation,differentiation and maturation of mineralization in vitro cultured osteoblasts of SD rats(OB). Methods The OB were isolated from neonatal SD rat calvaria in 24h by trypsin and collagenase digestion method. Set up 0,1,10,100 nmol·L-1,1α,25-(OH)2D3 as blank control,low,middle andhigh intervention group. Use the methyl thiazolyl tetrazolium assay(MTT)to detect the proliferation rate of OB. PNPP method was used for the determination of alkaline phosphatase(ALP)activity. Detect osteocalcin content(OC)by radioimmunoassay. The RT-PCR method was used to detect the expression of BMP-2 mRNA of osteoblast after intervening for 24,48 and 72h. Determinate calcium salt deposition after 3d and 6d;The number of cell calcified nodules was detected by alizarin red staining method after 10d and 20d. Results Compared with the 0 nmol/L group,the A value of osteoblast of 1 nmol/L group after intervening for 24h,48h and72h,respectively,increased by 20.50%, 38.77%,18.22%,and the differences were statistically significant(P<0.05);the ALP activity of osteoblasts of the 100 nmol/Lgroup were increased by 3.068±1.137,0.505±0.156,1.115±0.382,respectively,after the intervention of 24h,48h,72h,the OC content were increased by 0.285±0.097,0.469±0.134,0.734±0.225. The calcium depositions of osteoblast of 100 nmol/L group after the intervention of 3D and 6D,were increased by 0.968±0.236 and 0.929±0.307;Themineralization nodules of osteoblast of 100 nmol/L group were increased by 13.889±3.973 and 10.472±0.263after intervening for 10d and 20d,and there were significant statistical differences(P<0.05) andhad a positive correlation. Conclusion In the concentration range of this experiment,middle andhigh dose of 1α,25-(OH)2D3 can promote the differentiation andmineralization of osteoblast of newborn SD rats cultured in vitro,stimulate osteoblast secrete osteocalcin, plays the function of promoting bone formation.
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