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| 硫酸乙酰肝素对乳鼠颅骨成骨细胞增殖及分化成熟的影响 |
| Participation of heparin sulfate in fetal rat osteoblast cells proliferation, differentiation and mineralization |
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| DOI：10.3969/j.issn.1006.7108.2018.04.001 |
| 中文关键词: 硫酸乙酰肝素 成骨细胞 细胞培养 矿化 骨修复 |
| 英文关键词:Heparin sulfate Osteoblasts Cell culture Mineralization Bone repair |
| 基金项目:国家自然科学基金面上项目(21574059) ；国家自然科学基金青年科学基金资助项目( 81503007) |
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| 中文摘要: |
| 目的 探讨硫酸乙酰肝素（heparin sulfate, HS）对乳鼠颅骨成骨细胞增殖及分化成熟的影响。方法 酶消化法分离乳鼠颅骨的成骨细胞，培养并进行鉴定。取生长情况良好的成骨细胞制成浓度为2.0×104个/mL的细胞悬液，依据在成骨细胞培养基中添加不同浓度的HS，将实验分为A组（0 μg/mL）、B组（1 μg/mL）、C组（5 μg/mL）、D组（10 μg/mL）、E组（25 μg/mL）、F组（50 μg/mL）、G组（100 μg/mL）。以A为对照组，其余组为实验组。采用噻唑蓝（MTT）法检测48 h及96 h不同HS浓度下细胞的增殖情况，通过碱性磷酸酶（alkaline phosphatase, AKP）检测试剂盒测定细胞24 h和48 h的AKP活性，Western Blot法定量24 h和48 h成骨标志蛋白I型胶原蛋白（Collagen I）和Runx-2蛋白的表达，茜素红染色法计数14 d成骨细胞基质矿化结节量。结果 MTT检测示，硫酸乙酰肝素在48 h、96 h均抑制成骨细胞的增殖，相比于对照组差异有统计学意义（P<0.05）。AKP活性检测示，24 h和48 h的E组和F组的AKP活性相比于A组明显增高（P<0.05），且F组的AKP活性最高。Collagen I和Runx-2蛋白表达检测显示，50 μg/mL HS作用24 h和48 h后与对照组相比，Runx-2和Collagen I蛋白的表达均升高；100 μg/mL HS作用24 h和48 h后与对照组相比，Runx-2和Collagen I蛋白的表达均降低。矿化结节染色示，B、C、D、E、F组均能促进矿化结节的生成，F组对成骨细胞基质矿化作用最强，B、C、D、E和F组相比于A组差异均有统计学意义（P<0.05）。而G组的矿化结节数量低于A组。结论 HS的成骨作用显著，且50 μg/mL HS促进成骨细胞成熟分化的能力显著优于其他浓度。 |
| 英文摘要: |
| Objective To investigate the precise effect of Heparin sulfate (HS) on the proliferation, differentiation and mineralization of osteoblasts in vitro. Methods Osteoblasts were isolated from neonatal rat calvaria with enzyme digestion, and HS experimental concentrations were divided into 7 groups (A (0 μg /mL), B (1 μg /mL), C (5 μg /mL), D (10 μg /mL), E (25 μg /mL), F (50 μg /mL) and G (100 μg /mL)). Well-growing osteoblasts were cultured with different HS concentrations in the medium of 2×104 /mL cell suspension. MTT assay was used to detect the cells proliferation at 48h and 96h under different HS concentrations. The AKP activities at 24 h and 48 h were determined by alkaline phosphatase assay kit, quantitative expression of osteogenic markers Collagen I and Runx-2 protein was measured by Western Blot, and the mineralization rates were calculated by alizarin red staining method in 14 days. Results MTT assay showed that HS inhibited the proliferation of osteoblasts at 48 h and 96 h, compared with the control group (P<0.05), which had statistical significance. AKP activity test showed that the osteogenic activities of group E and group F were significantly higher than those in group A at 24 h and 48 h (P<0.05), and the AKP activity of group F was the highest. The Western Blot assay showed that, compared with the control group, the expression of Runx-2 and Collagen I protein increased in the 50 μg/mL HS group at 24 h and 48 h (P<0.05), but decreased in the 100 μg /mL HS group. Mineralized nodules staining showed that different concentrations of HS could promote the formation of mineralized nodules except in group G, group F had the strongest mineralization effect on osteoblasts matrix, and the differences were statistically significant between group A and other effective groups (P<0.05). Conclusion Osteogenic effect of HS is remarkable, and 50 μg/mL HS is the best concentration for the maturation and differentiation of osteoblasts, significantly better than other concentrations. |
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