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• W4283721714 abstract "Objective: 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 expressions of heat shock protein 90 (HSP90), caveolin 1, endothelial nitric oxide synthase (eNOS), and eNOS phosphorylation site 1177 in the cells, and the eNOS phosphorylation site 1177/eNOS ratio was calculated, with the number of samples being 3; co-immunoprecipitation (co-precipitating HSP90 and caveolin 1, caveolin 1 and eNOS) and Western blotting were used to detect the protein expressions of caveolin 1 and eNOS in the cells, with the number of samples being 3; the protein co-localization of HSP90 and caveolin 1 and that of caveolin 1 and eNOS in the cells was assessed by immunofluorescence double staining. The molecular docking prediction of caveolin 1 and eNOS was processed by HADDOCK 2.4 protein-protein docking program. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and least significant difference method. Results: Compared with that in normal control group, the cell proliferation level in 17-AAG alone group was significantly decreased at culture hour of 24, 48, and 72 after the treatment (P<0.01), while the cell proliferation level in negative pressure treatment alone group was significantly increased at culture hour of 24, 48, and 72 after the treatment (P<0.01). Compared with that in 17-AAG alone group, the cell proliferation level in 17-AAG+negative pressure treatment group was significantly increased at culture hour of 48 and 72 after the treatment (P<0.05 or P<0.01). Compared with that in negative pressure treatment alone group, the cell proliferation level in 17-AAG+negative pressure treatment group was significantly decreased at culture hour of 24, 48, and 72 after the treatment (P<0.01). At 12 h after scratching, compared with (39.9±2.7)% in normal control group, the cell migration rate in 17-AAG alone group was significantly decreased ((10.7±2.7)%, P<0.01), while the cell migration rate in negative pressure treatment alone group was significantly increased ((61.9±2.4)%, P<0.01). Compared with those in 17-AAG alone group, the cell migration rate in 17-AAG+negative pressure treatment group was significantly increased ((37.7±3.7)%, P<0.01). Compared with that in negative pressure treatment alone group, the cell migration rate in 17-AAG+negative pressure treatment group was significantly decreased (P<0.01). At culture hour of 6 after the treatment, compared with those in normal control group, the total length of the tube formed by the cells in 17-AAG alone group was significantly shortened (P<0.05) and the number of branch nodes was significantly reduced (P<0.05), while the total length of the tube formed by the cells in negative pressure treatment alone group was significantly prolonged (P<0.01) and the number of branch nodes was dramatically increased (P<0.01). Compared with that in 17-AAG alone group, the number of branch nodes of the tube formed by the cells was significantly increased in 17-AAG+negative pressure treatment group (P<0.05). Compared with those in negative pressure treatment alone group, the total length of the tube formed by the cells in 17-AAG+negative pressure treatment group was significantly shortened (P<0.01) and the number of branch nodes was significantly reduced (P<0.01). Western blotting detection showed that after treatment, the overall comparison of eNOS and caveolin 1 protein expressions among the three groups of cells showed no statistically significant differences (P>0.05). The expression of HSP90 protein and the eNOS phosphorylation site 1177/eNOS ratio in the cells of negative pressure treatment alone group were significantly increased (P<0.01) compared with those in normal control group. Compared with those in negative pressure treatment alone group, the HSP90 protein expression and the eNOS phosphorylation site 1177/eNOS ratio in the cells of 17-AAG+negative pressure treatment group were significantly decreased (P<0.01). Co-immunoprecipitation and Western blotting detection after the treatment showed that compared with those in normal control group, the expression of caveolin 1 protein in the cells of negative pressure treatment alone group was significantly increased (P<0.01), while the protein expression of eNOS was significantly decreased (P<0.05). Compared with those in negative pressure treatment alone group, the expression of caveolin 1 protein in the cells of 17-AAG+negative pressure treatment group was significantly decreased (P<0.01), while the protein expression of eNOS was significantly increased (P<0.01). After the treatment, compared with those in normal control group, the co-localization of HSP90 and caveolin 1 protein in the cells of negative pressure treatment alone group was significantly increased, while the co-localization of caveolin 1 and eNOS protein was significantly decreased. Compared with those in negative pressure treatment alone group, the co-localization of HSP90 and caveolin 1 protein in the cells of 17-AAG+negative pressure treatment group was significantly decreased, while the co-localization of caveolin 1 and eNOS protein was significantly increased. Molecular docking prediction suggested that caveolin 1 interacted strongly with eNOS and inhibited the 1177 site phosphorylation of eNOS. Conclusions: The negative pressure microenvironment may inhibit the binding of caveolin 1 to eNOS by promoting the binding of HSP90 to caveolin 1 in HUVECs, so as to relieve the inhibition of 1177 site phosphorylation of eNOS by caveolin 1, thereby promoting the proliferation, migration, and tubulogenesis of HUVECs, and ultimately promoting the neogenesis of HUVECs.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、72 h均明显升高（P<0.01）；与单纯17-AAG组相比，17-AAG+负压处理组细胞增殖水平于处理完成后培养48、72 h均明显升高（P<0.05或P<0.01）；与单纯负压处理组相比，17-AAG+负压处理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）。划痕后12 h，与正常对照组的（39.9±2.7）%相比，单纯17-AAG组细胞迁移率明显降低［（10.7±2.7）%，P<0.01］，单纯负压处理组细胞迁移率明显升高［（61.9±2.4）%，P<0.01］；与单纯17-AAG组相比，17-AAG+负压处理组细胞迁移率明显升高［（37.7±3.7）%，P<0.01］；与单纯负压处理组相比，17-AAG+负压处理组细胞迁移率明显降低（P<0.01）。处理完成后培养6 h，与正常对照组相比，单纯17-AAG组细胞成管总长度明显缩短（P<0.05）且分支节点数明显减少（P<0.05），单纯负压处理组细胞成管总长度明显延长（P<0.01）且分支节点数明显增加（P<0.01）；与单纯17-AAG组相比，17-AAG+负压处理组细胞成管分支节点数明显增加（P<0.05）；与单纯负压处理组相比，17-AAG+负压处理组细胞成管总长度明显缩短（P<0.01）且分支节点数明显减少（P<0.01）。蛋白质印迹法检测显示，处理完成后，3组细胞中eNOS、窖蛋白1蛋白表达总体比较，差异均无统计学意义（P>0.05）；单纯负压处理组细胞中HSP90蛋白表达与eNOS磷酸化位点1177/eNOS比值均明显高于正常对照组（P<0.01），17-AAG+负压处理组细胞中HSP90蛋白表达与eNOS磷酸化位点1177/eNOS比值均明显低于单纯负压处理组（P<0.01）。处理完成后免疫共沉淀与蛋白质印迹法检测显示，与正常对照组相比，单纯负压处理组细胞中窖蛋白1蛋白表达明显升高（P<0.01），eNOS蛋白表达明显降低（P<0.05）；与单纯负压处理组相比，17-AAG+负压处理组处理完成后细胞中窖蛋白1蛋白表达明显降低（P<0.01），eNOS蛋白表达明显升高（P<0.01）。处理完成后，与正常对照组相比，单纯负压处理组细胞中HSP90与窖蛋白1的蛋白共定位明显增多，窖蛋白1与eNOS的蛋白共定位明显减少；与单纯负压处理组比，17-AAG+负压处理组细胞中HSP90与窖蛋白1的蛋白共定位明显减少，窖蛋白1与eNOS的蛋白共定位明显增多。分子对接预测提示，窖蛋白1与eNOS相互作用较强，抑制eNOS 1177位点的磷酸化。 结论： 负压微环境可能通过促进HUVEC中HSP90结合窖蛋白1进而抑制窖蛋白1结合eNOS，以解除窖蛋白1对eNOS 1177位点磷酸化的抑制，从而促进HUVEC增殖、迁移和成管，最终促进HUVEC新生。." @default.
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• W4283721714 date "2022-06-20" @default.
• W4283721714 modified "2023-09-28" @default.
• W4283721714 title "[Effects and mechanism of negative pressure microenvironment on the neogenesis of human umbilical vein endothelial cells]." @default.
• W4283721714 doi "https://doi.org/10.3760/cma.j.cn501225-20220119-00009" @default.
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