负压微环境对人脐静脉血管内皮细胞新生的影响及其机制  

Effects and mechanism of negative pressure microenvironment on the neogenesis of human umbilical vein endothelial cells

作　　者：董禹辰 黄容 赵聪颖 李学拥 Yuchen Dong;Rong Huang;Congying Zhao;Xueyong Li(Department of Burns and Plastic Surgery,the Second Affiliated Hospital of Air Force Medical University,Xi'an 710038,China)

机构地区：[1]空军军医大学第二附属医院烧伤整形科,西安710038

出　　处：《中华烧伤与创面修复杂志》2022年第6期520-531,共12页Chinese Journal of Burns And Wounds

基　　金：陕西省重点研发计划(2020ZDLSF04-13,2021SF-292);空军军医大学军事医学“珠峰工程”项目(2020ZFC004);空军军医大学第二附属医院学科提升平台项目(2020XKPT013)。

摘　　要：目的探究负压微环境对人脐静脉血管内皮细胞(HUVEC)新生的影响及其机制。方法采用实验研究方法。取第3~5代对数生长期HUVEC进行后续实验。取3批细胞,各批次细胞均分为常规培养24 h的正常对照组和单纯负压处理组以及加入17-丙烯胺基-17-去甲氨基格尔德霉素(17-AAG)培养24 h的单纯17-AAG组与17-AAG+负压处理组,另采用自行设计研发的全自动三维细胞梯度负压加载装置对2个负压处理组细胞行持续8 h的间歇负压吸引(负压值为-5.33 kPa,吸引30 s、暂停10 s),第1个批次细胞处理完成后于培养0(即刻)、24、48、72 h,采用细胞计数试剂盒8法检测细胞增殖水平,样本数为6;第2个批次细胞处理完成后进行划痕试验,于划痕后12 h,在倒置相差显微镜下观察细胞迁移情况后计算细胞迁移率,样本数为3;第3个批次细胞处理完成后进行小管形成实验,于培养6 h,在倒置相差显微镜下观察成管情况后计算细胞成管总长度与分支节点数,样本数为3。取细胞,分为正常对照组、单纯负压处理组、17-AAG+负压处理组,同前相应组别进行处理,处理完成后,采用蛋白质印迹法检测细胞中热休克蛋白90(HSP90)、窖蛋白1、内皮型一氧化氮合酶(eNOS)、eNOS磷酸化位点1177蛋白表达并计算eNOS磷酸化位点1177/eNOS比值(样本数为3),采用免疫共沉淀(共沉淀HSP90与窖蛋白1、窖蛋白1与eNOS)与蛋白质印迹法检测各组细胞中窖蛋白1、eNOS的蛋白表达(样本数为3),采用免疫荧光双重染色法评估细胞中HSP90与窖蛋白1、窖蛋白1与eNOS的蛋白共定位情况。通过HADDOCK 2.4蛋白质-蛋白质对接程序对窖蛋白1和eNOS进行分子对接预测。数据分析采用析因设计方差分析、单因素方差分析、LSD法。结果与正常对照组相比,单纯17-AAG组细胞增殖水平于处理完成后培养24、48、72 h均明显降低(P<0.01),单纯负压处理组细胞增殖水平于处理完成后培养24、48、7Objective To investigate the effects and mechanism of negative pressure microenvironment on the neogenesis of human umbilical vein endothelial cells(HUVECs).Methods The experimental research methods were adopted.The third to the fifth passage of HUVECs in the logarithmic growth stage were used for the subsequent experiments.Three batches of cells were taken,with each batch of cells being divided into normal control group and negative pressure treatment alone group(both routinely cultured for 24 h),and 17-allylamino-17-demethoxy-geldanamycin(17-AAG)alone group and 17-AAG+negative pressure treatment group(both cultured with 17-AAG for 24 h).In addition,the intermittent negative pressure suction,with the negative pressure value of-5.33 kPa(suction for 30 s,pause for 10 s)was continuously applied for 8 h on cells in the two negative pressure treatment groups using an automatic three-dimensional cell gradient negative pressure loading device designed and developed by ourselves.After the treatment of the first batch of cells,the cell proliferation level was detected by cell counting kit 8 method at 0(immediately),24,48,and 72 h of culture,with the number of samples being 6.After the treatment of the second batch of cells,the scratch experiment was performed.At 12 h after scratching,the cell migration was observed under an inverted phase contrast microscope and the cell migration rate was calculated,with the number of samples being 3.After the treatment of the third batch of cells,the tubule formation experiment was conducted.After 6 h of culture,the tubulogenesis was observed under an inverted phase contrast microscope and the total tubule length and the number of branch nodes of cells were calculated,with the number of samples being 3.The cells were taken and divided into normal control group,negative pressure treatment alone group,and 17-AAG+negative pressure treatment group.The cells were treated the same as in the previous corresponding group.After the treatment,Western blotting was used to detect the protein expre

关 键 词：负压伤口疗法 细胞微环境 HSP90热休克蛋白质类 窖蛋白1 血管内皮细胞 内皮型一氧化氮合酶 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]