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Title: [Effect of natural hirudin on angiogenesis of human microvascular endothelial cells].
Author: Lin G, Lin B, Zhu J, Pan X, Yin G.
Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2018 Dec 15; 32(12):1586-1591. PubMed ID: 30569688.
Abstract:
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), and A 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. 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。.

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