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| 维生素D3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 D3 and its metabolites on the differentiation and biomineralization of osteoblasts in vitro |
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| DOI：10.3969/j.issn.1006-7108.2021.03.007 |
| 中文关键词: 维生素D3 25-羟基维生素D3 1α,25-二羟基维生素D3 成骨细胞 分化 生物矿化 |
| 英文关键词:vitamin D3 25-Hydroxyvitamin D3 1α,25-dihydroxyvitamin D3 osteoblast differentiation biomineralization |
| 基金项目:航天医学基础与应用国家重点实验室开放基金（SMFA13K02） |
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| 中文摘要: |
| 目的 研究体外维生素D3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VD3、25(OH)VD3和1α,25(OH)2VD3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VD3及其代谢物的反应，并对成骨标志物进行分析，评价VD3及其代谢物对成骨细胞分化和生物矿化能力的影响。结果 VD3及其代谢物对成骨细胞无细胞毒性，只有200 nmol/L的VD3能显著促进成骨细胞增殖，而25(OH)VD3和1α,25(OH)2VD3对成骨细胞增殖的促进作用不明显。25(OH)VD3和1α,25(OH)2VD3可以上调成骨细胞CYP24A1基因的转录活性，但不能直接代谢VD3和25(OH)VD3。25(OH)VD3和1α,25(OH)2VD3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)VD3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)VD3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。 |
| 英文摘要: |
| Objective To study the effect of VD3 and its metabolites on the osteoblast differentiation and biomineralization in different concentrations. Methods Osteoblasts were isolated with collagenase digestion method. The control group, VD3 treatment group, 25(OH)VD3 treatment group, and 1α,25(OH)2VD3 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 VD3, 25(OH)VD3, or 1α,25(OH)2VD3. The effects of VD3 and its metabolites on the differentiation and biomineralization of osteoblasts were evaluated with osteoblast marker analysis. Results The experimental results confirmed that VD3 and its metabolites had no cytotoxicity to osteoblasts. The proliferation of osteoblasts was promoted significantly with 200 nmol/L of VD3 only. It was not promoted significantly with 25(OH)VD3 and 1α,25(OH)2VD3. Moreover, 25(OH)VD3 and 1α,25(OH)2VD3 up-regulated the transcription activity of CYP24A1 gene in osteoblasts, but did not metabolize VD3 and 25(OH)VD3 directly. The expression of osteogenic markers (Runx2, ALP, etc.) was promoted by 25(OH)VD3 and 1α,25(OH)2VD3 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)VD3 only. Conclusion In vitro, 25(OH)VD3 induces osteoblast differentiation and biomineralization, which can provide evidence for its application in clinical osteoporosis and bone tissue engineering. |
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