水蛭素通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化

摘要：目的 观察水蛭素对人骨髓间充质干细胞（bone marrow mesenchymal stem cell，BMSC）成骨分化的影响。方法 BMSCs细胞分为正常培养的对照组、成骨诱导的诱导组以及加入不同浓度（1、10、20 ATU/mL）处理的水蛭素组。MTT检测细胞增值并筛选水蛭素最适作用浓度。流式细胞仪检测细胞凋亡。RT-PCR和Western blot分别检测成骨基因Runx2、Osterix、COL1A1的mRNA和蛋白表达。BCIP/NBT染色法检测细胞中的碱性磷酸酶水平。茜素红染色检测矿化结节。检测VEGF、Notch1、Jagged1和CBF1的mRNA和蛋白表达。结果 骨髓间充质干细胞经成骨诱导细胞增殖显著增加，中高浓度的水蛭素可以不同程度促进成骨诱导的BMSCs细胞增殖（P＜0.05），并筛选出20 ATU/mL作为水蛭素的使用浓度。水蛭素抑制成骨诱导的BMSCs细胞凋亡，上调Runx2、Osterix、COL1A1的mRNA和蛋白表达，增加碱性磷酸酶水平，促进细胞中矿化结节的生成，并提升BMSCs细胞中VEGF、Notch1、Jagged1和CBF1的表达（P＜0.05）。结论 水蛭素可能通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化。

Hirudin promotes osteogenic differentiation of bone marrow mesenchymal stem cells by up-regulating VEGF/Notch1 signaling pathway

Abstract: Objective To observe the effect of hirudin on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods BMSCs cells were divided into normal cultured control group, osteogenic induced group, and different hirudin groups (1, 10, 20 ATU/mL). MTT method was used to detect cell proliferation and screen the optimal concentration of hirudin. Cell apoptosis was measured using flow cytometry method. mRNA and protein expressions of Runx2, Osterix, and COL1A1 were measured using RT-PCR and Western blotting, respectively. Alkaline phosphatase was detected with BCIP/NBT staining. Alizarin red staining was used to detect mineralized nodules. mRNA and protein expressions of VEGF, Notch1, Jagged1, and CBF1 were measured. Results BMSCs cell proliferation increased significantly after osteogenic induction. Hirudin at medium/high concentration elevated cell proliferation of osteogenic induced BMSCs (P<0.05), and 20 ATU/mL was selected as the optimal concentration of hirudin. Hirudin decreased cell apoptosis, elevated mRNA and protein expression level of Runx2, Osterix, and COL1A1, increased alkaline phosphatase levels, promoted the formation of mineralized nodules, and increased expressions of VEGF, Notch1, Jagged1, and CBF1 in osteogenic induced BMSCs (P<0.05). Conclusion Hirudin may promote osteogenic differentiation of bone marrow mesenchymal stem cells by up-regulating VEGF/Notch1 signaling pathway.