中国修复重建外科杂志

中国修复重建外科杂志

天然水蛭素对人微血管再生作用的初步研究

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目的 研究天然水蛭素对人微血管内皮细胞(human microvascular endothelial cells,HMVECs)增殖的影响及其诱导血管再生的机制。 方法 取 HMVECs 接种至 Matrigel 基质胶培养 24 h,体外建立三维培养模型,于倒置相差显微镜下观察细胞生长情况。然后将细胞三维培养模型随机分为不同浓度天然水蛭素培养组(1、4、7 ATU/mL 组)以及单纯含 10%FBS 的 DMEM 培养基培养组(对照组)。各组细胞三维培养模型加入对应培养基后,培养 24、48、72 h 采用细胞计数试剂盒 8(cell counting kit 8,CCK-8)检测细胞增殖情况;培养 24 h 行成管实验并采用免疫荧光染色法测定细胞 VEGF 及 Notch1 表达水平。 结果 细胞三维培养观察示,HMVECs 于基质胶上贴附良好,24 h 时完全形成管腔样结构。CCK-8 检测示,各时间点 1、4 ATU/mL 组 A 值均高于对照组(P<0.05),其中 4 ATU/mL 组最高;而 7 ATU/mL 组A 值显著低于对照组以及 1、4 ATU/mL 组(P<0.05)。细胞成管实验示,1、4 ATU/mL 组成管数量较对照组及 7 ATU/mL 组明显增多,且 4 ATU/mL 组多于 1 ATU/mL 组,差异有统计学意义(P<0.05);7 ATU/mL 组与对照组比较,差异无统计学意义(P>0.05)。免疫荧光染色示,与对照组比较,1、4 ATU/mL 组 Notch1 表达量增高,7 ATU/mL 组降低;其中 4、7 ATU/mL 组与对照组比较差异有统计学意义(P<0.05)。1、4、7 ATU/mL 组 VEGF 表达量均较对照组增高,4 ATU/mL 组高于 1、7 ATU/mL 组,差异均有统计学意义(P<0.05)。 结论 低、中浓度天然水蛭素可促进 HMVECs 增殖使血管再生,高浓度天然水蛭素则对 HMVECs 增殖及血管再生起抑制作用;其作用机制可能与 VEGF-Notch 信号通路有关。

Objective To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. Results The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance (A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point (P<0.05), andA value of 4 ATU/mL group was the highest. The A value of 7 ATU/mL group was significantly lower than those of 1 and 4 ATU/mL groups and control group (P<0.05). The tube formation assay showed that the tube structure was more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in 1 ATU/mL group, showing significant differences (P<0.05). There was no significant difference between 7 ATU/mL group and control group (P>0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group (P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences (P<0.05). Conclusion Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.

关键词: 水蛭素; 微血管内皮细胞; 三维培养; Notch 信号通路; 血管再生

Key words: Hirudin; microvascular endothelial cells; three-dimensional culture; Notch signaling pathway; angiogenesis

引用本文: 林冠宇, 林博杰, 朱江英, 潘新元, 殷国前. 天然水蛭素对人微血管再生作用的初步研究. 中国修复重建外科杂志, 2018, 32(12): 1586-1591. doi: 10.7507/1002-1892.201806055 复制

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