摘要
目的 观察1,25-二羟维生素D3[1,25(OH)2D3]对体外培养的小鼠成骨细胞osteoprotegerin(OPG)和receptor activator of NF-κB ligand(RANKL)基因表达的作用。方法 取30只出生24 h内的新生小鼠,在无菌条件下取颅盖骨,去除纤维结缔组织,用胰蛋白酶-胶原酶消化法进行成骨细胞的原代培养。取第3～5代的成骨细胞,分成对照组和实验组,在实验组培养液中加入不同浓度的1,25(OH)2D3(10-8、10-9、10-11mol/L),培养48 h后,取出培养液,-70℃冻存备用。从培养瓶中贴壁细胞提取总RNA,应用RT-PCR法检测细胞中OPG和RANKL的mRNA表达。应用ELISA方法检测培养液中OPG蛋白的浓度。结果 各浓度1,25(OH)2D3作用的成骨细胞中,OPG的mRNA表达均较对照组显著减弱,并随1,25(OH)2D3浓度的增加而表达减弱,呈现明显的浓度依赖性;实验组RANKL mRNA的表达较对照组表达明显,且随1,25(OH)2D3浓度增加而增强;实验组成骨细胞中OPG蛋白的表达明显低于对照组(P<0.05)。结论 1,25(OH)2D3抑制小鼠成骨细胞OPG的表达,同时增加RANKL的表达。
Objective To study the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of osteoprotegerin (OPG) and receptor activator of NF-KB ligand (RANKL) mRNA in mouse osteoblasts. Methods Calvariae derived from CD-I neonatal mouse (after born 24 h). Bone samples were processed by the collagenase/trypsin digestion method. Mouse osteoblasts were cultured in vitro. After 48 hours of addition of 1,25(OH)2D3 (0, 10-8, 10-9, 10-11 mol/L) to the culture medium of mouse osteoblasts, the content of the OPG protein in culture medium was estimated with enzyme linked immunosorbent assay. Total RNA was prepared from mouse osteoblasts. mRNA expression of OPG and RANKL were detected by reverse transcription-polymerase chain reaction. Results The mRNA expression of OPG in osteoblasts added with 1,25(OH)2D3 significantly decreased compared with the controls, which was markedly dose-dependent. OPG protein production in the medium decreased after treatment with 1,25(OH)2D3. In contrast, RANKL mRNA expression levels in osteoblasts significantly increased after 48 h of culture with 1,25(OH)2D3. Conclusion 1,25 (OH)2D3 can stimulate RANKL mRNA expression, but decrease OPG mRNA levels in vitro in mouse osteoblasts.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2004年第4期418-422,共5页
Acta Academiae Medicinae Sinicae
关键词
OPG
RANKL
1
25-二羟维生素D3
成骨细胞
osteoprotegerin
receptor activator of NF-κB ligand
1,25-dihydroxyvitamin D3
osteoblast
