维生素D_(3)及其代谢物对成骨细胞分化及生物矿化的实验研究

Experimental study on the effect of vitamin D_(3) and its metabolites on the differentiation and biomineralization of osteoblasts in vitro

目的研究体外维生素D_(3)及其代谢物在不同浓度下对成骨细胞分化和生物矿化的影响。方法采用胶原酶消化法分离成骨细胞;设立对照组,不同浓度VD_(3)、25(OH)VD_(3)和1α,25(OH)2VD_(3)药物处理组。采用CCK-8法检测细胞增殖,采用PNPP法测定ALP活性,采用钙含量测定、ELISA法和实时定量聚合酶链反应来评估成骨细胞对VD_(3)及其代谢物的反应,并对成骨标志物进行分析,评价VD_(3)及其代谢物对成骨细胞分化和生物矿化能力的影响。结果VD_(3)及其代谢物对成骨细胞无细胞毒性,只有200 nmol/L的VD_(3)能显著促进成骨细胞增殖,而25(OH)VD_(3)和1α,25(OH)2VD_(3)对成骨细胞增殖的促进作用不明显。25(OH)VD_(3)和1α,25(OH)2VD_(3)可以上调成骨细胞CYP24A1基因的转录活性,但不能直接代谢VD_(3)和25(OH)VD_(3)。25(OH)VD_(3)和1α,25(OH)2VD_(3)以剂量依赖性增加的方式促进早期成骨标志物(Runx2,ALP等)的表达。只有25(OH)VD_(3)能促进成骨细胞OCN基因和蛋白的表达,提高成骨细胞的生物矿化水平。结论体外25(OH)VD_(3)可诱导成骨细胞的分化和生物矿化,为其在临床骨质疏松症和骨组织工程中的应用提供依据。

Objective To study the effect of VD_(3) and its metabolites on the osteoblast differentiation and biomineralization in different concentrations.Methods Osteoblasts were isolated with collagenase digestion method.The control group,VD_(3) treatment group,25(OH)VD_(3) treatment group,and 1α,25(OH)2VD_(3) treatment group were established,respectively.CCK-8 test was used to detect the proliferation rate of osteoblasts.PNPP method was used for the determination of alkaline phosphatase activity.Calcium content determination,ELISA,and real-time quantitative polymerase chain reaction were used to evaluate the response of osteoblasts to VD_(3),25(OH)VD_(3),or 1α,25(OH)2VD_(3).The effects of VD_(3) and its metabolites on the differentiation and biomineralization of osteoblasts were evaluated with osteoblast marker analysis.Results The experimental result confirmed that VD_(3) and its metabolites had no cytotoxicity to osteoblasts.The proliferation of osteoblasts was promoted significantly with 200 nmol/L of VD_(3) only.It was not promoted significantly with 25(OH)VD_(3) and 1α,25(OH)2VD_(3).Moreover,25(OH)VD_(3) and 1α,25(OH)2VD_(3) up-regulated the transcription activity of CYP24A1 gene in osteoblasts,but did not metabolize VD_(3) and 25(OH)VD_(3) directly.The expression of osteogenic markers(Runx2,ALP,etc.)was promoted by 25(OH)VD_(3) and 1α,25(OH)2VD_(3) in a dose-dependent manner.More importantly,the gene and protein expressions of osteocalcin and the biomineralization level of osteoblasts were promoted by 25(OH)VD_(3) only.Conclusion In vitro,25(OH)VD_(3) induces osteoblast differentiation and biomineralization,which can provide evidence for its application in clinical osteoporosis and bone tissue engineering.