天然水蛭素对人微血管再生作用的初步研究  

Effect of natural hirudin on angiogenesis of human microvascular endothelial cells

作　　者：林冠宇 林博杰[1] 朱江英[1] 潘新元 殷国前[1] LIN Guanyu;LIN Bojie;ZHU Jiangying;PAN Xinyuan;YIN Guoqian(Department of Plastic and Aesthetic Surgery,the First Affiliated Hospital of Guangxi Medical University,Nanning Guangxi,530021,P.R.China;Department of Burn and Plastic Surgery,Minzu Hospital of Zhuang Autonomous Region,Nanning Guangxi,530001,P.R.China)

机构地区：[1]广西医科大学第一附属医院整形美容外科,南宁530021 [2]广西壮族自治区民族医院烧伤整形外科,南宁530001

出　　处：《中国修复重建外科杂志》2018年第12期1586-1591,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81460297)~~

摘　　要：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 microvascularendothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods Three-dimensionalculture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hoursof 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% fetalbovine 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 ofvascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescencestaining at 24 hours. Results The observation of cells in three-dimensional culture models showed that HMVECsattached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showedthat 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 thanthose of 1 and 4 ATU/mL groups and control group (P<0.05). The tube formation assay showed that the tube structurewas more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in1 ATU/mL group, showing significant differences (P<0.05). There was no significant difference between 7 ATU/mL groupand control group (P>0.05). The results of immunofluorescence staining showed that compared with control group, theNotch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significantdifference between 4 and 7 ATU/mL groups and control group (P<0.05). The VEGF expression was higher in 1, 4, and7 ATU/mL groups than in control g

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 

分 类 号：R285[医药卫生—中药学]