Effects and mechanism of negative pressure microenvironment on the neogenesis of human umbilical vein endothelial cells
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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、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。 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. -
参考文献
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1 全自动三维细胞梯度负压加载装置的设计与实物及应用。1A.结构示意图,其中左图为正视图，右图为侧后视图；1B.装置实物图，图中上半部分与下半部分分别是梯度负压调控组件与智能控制系统组件；1C.BioFlex培养板放置过程图；1D.激光测距装置发射光提示负压泵对刺激腔持续施加负压
2 4组人脐静脉血管内皮细胞处理完成后划痕后各时间点细胞划痕面积情况 倒置相差显微镜×100，图中标尺为200 μm。2A、2B、2C、2D.分别为正常对照组、单纯17-丙烯胺基-17-去甲氨基格尔德霉素（17-AAG）组、单纯负压处理组、17-AAG+负压处理组划痕后0 h（即刻），划痕情况相近；2E、2F、2G、2H.分别为正常对照组、单纯17-AAG组、单纯负压处理组、17-AAG+负压处理组划痕后12 h，图2E划痕面积较大，图2F划痕面积明显大于图2E，图2G划痕面积明显小于图2E，图2H划痕面积明显小于图2F但明显大于图2G
3 4组人脐静脉血管内皮细胞处理完成后培养6 h成管情况 倒置相差显微镜×100，图中标尺为200 μm。3A、3B、3C、3D.分别为正常对照组、单纯17-丙烯胺基-17-去甲氨基格尔德霉素（17-AAG）组、单纯负压处理组、17-AAG+负压处理组，与图3A相比，图3B中细胞成管总长度明显缩短且分支节点数明显减少，图3C中细胞成管总长度明显延长且分支节点数明显增加；与图3B相比，图3D中细胞成管总长度无明显延长但分支节点数明显增加；与图3C相比，图3D中细胞成管总长度明显缩短且分支节点数明显减少
4 蛋白质印迹法检测3组人脐静脉血管内皮细胞处理完成后细胞中的eNOS磷酸化位点1177、eNOS、HSP90、窖蛋白1蛋白表达
注：eNOS为内皮型一氧化氮合酶，HSP90为热休克蛋白90，GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2、3分别指正常对照组、单纯负压处理组、17-丙烯胺基-17-去甲氨基格尔德霉素+负压处理组
5 免疫共沉淀与蛋白质印迹法检测3组人脐静脉血管内皮细胞和阴性对照处理完成后细胞中窖蛋白1与eNOS蛋白表达。5A.窖蛋白1；5B.eNOS
注：HSP90为热休克蛋白90，eNOS为内皮型一氧化氮合酶；条带上方1、2、3、4分别指正常对照组、单纯负压处理组、17-丙烯胺基-17-去甲氨基格尔德霉素+负压处理组、阴性对照
6 3组人脐静脉血管内皮细胞处理完成后细胞中HSP90与窖蛋白1的蛋白表达 花青素3-Alexa Fluor 488-4′,6-二脒基-2-苯基吲哚×200，图中标尺为50 μm。6A、6B、6C、6D与6E、6F、6G、6H及6I、6J、6K、6L.分别为正常对照组与单纯负压处理组及17-丙烯胺基-17-去甲氨基格尔德霉素+负压处理组HSP90染色、窖蛋白1染色、细胞核染色、细胞核与HSP90及窖蛋白1染色重叠图片，图6A中HSP90表达少，图6B中窖蛋白1表达明显，图6C中细胞核完整，图6D中HSP90与窖蛋白1的蛋白共定位较少；图6E中HSP90表达较图6A多，图6F中窖蛋白1表达与图6B相近，图6G中细胞核完整，图6H中HSP90与窖蛋白1的蛋白共定位较图6D多；图6I中HSP90表达较图6E少，图6J中窖蛋白1表达与图6F相近，图6K中细胞核完整，图6L中HSP90与窖蛋白1的蛋白共定位较图6H少
注：热休克蛋白90（HSP90）阳性染色为红色，窖蛋白1阳性染色为绿色，细胞核阳性染色为蓝色，蛋白共定位染色为黄色
7 3组人脐静脉血管内皮细胞处理完成后细胞中eNOS与窖蛋白1的蛋白表达 花青素 3-Alexa Fluor 488-4′,6-二脒基-2-苯基吲哚×200，图中标尺为50 μm。7A、7B、7C、7D与7E、7F、7G、7H及7I、7J、7K、7L.分别为正常对照组与单纯负压处理组及17-丙烯胺基-17-去甲氨基格尔德霉素+负压处理组eNOS染色、窖蛋白1染色、细胞核染色、细胞核与eNOS及窖蛋白1染色重叠图片，图7A中eNOS表达明显，图7B中窖蛋白1表达明显，图7C中细胞核完整，图7D中eNOS和窖蛋白1的蛋白共定位多；图7E中eNOS表达较图7A减少，图7F中窖蛋白1表达较图7B减少，图7G中细胞核完整，图7H中eNOS和窖蛋白1的蛋白共定位较图7D减少；图7I中eNOS表达较图7E增多，图7J中窖蛋白1表达较图7F增多，图7K中细胞核完整，图7L中eNOS和窖蛋白1的蛋白共定位较图7H增多
注：内皮型一氧化氮合酶（eNOS）阳性染色为红色，窖蛋白1阳性染色为绿色，细胞核阳性染色为蓝色，蛋白共定位染色为黄色
8 通过HADDOCK 2.4蛋白质-蛋白质对接程序预测生成的窖蛋白1与eNOS的分子对接最佳结合姿势图。8A.2个分子对接完整图；8B.8A中方框区域放大图
注：左侧浅蓝色区域为内皮型一氧化氮合酶（eNOS）分子结构，右侧翠绿色区域为窖蛋白1分子结构；蓝色、深绿色树枝形状表示窖蛋白1与eNOS结合的各氨基酸位点，序号为2个分子对接的主要位点；字母表示各氨基酸，如SER表示丝氨酸、HIS表示组氨酸、GLY表示甘氨酸、LEU表示异亮氨酸、GLN表示谷氨酰胺等；丝氨酸1177位点位于对接界面中，形成了阻碍其他分子接触该氨基酸的空间构象
表1 4组人脐静脉血管内皮细胞处理完成后培养各时间点增殖水平比较(
组别 样本数 0 h（即刻） 24 h 48 h 72 h 正常对照组 6 0.251±0.032 0.401±0.051 0.512±0.072 0.902±0.081 单纯17-AAG组 6 0.222±0.031 0.332±0.031 0.393±0.043 0.713±0.040 单纯负压处理组 6 0.243±0.022 0.513±0.033 0.932±0.041 1.402±0.052 17-AAG+负压处理组 6 0.252±0.032 0.362±0.042 0.561±0.073 0.802±0.073 F值 0.97 25.35 104.15 141.74 P值 0.428 <0.001 <0.001 <0.001 P1值 — 0.006 <0.001 <0.001 P2值 — <0.001 <0.001 <0.001 P3值 — 0.214 <0.001 0.019 P4值 — <0.001 <0.001 <0.001 注：处理因素主效应，F=243.98，P<0.001;时间因素主效应，F=963.65，P<0.001；两者交互作用，F=50.10，P<0.001；F值、P值为4组间各时间点总体比较所得；P1值为单纯17-丙烯胺基-17-去甲氨基格尔德霉素（17-AAG）组与正常对照组各时间点比较所得，P2值为单纯负压处理组与正常对照组各时间点比较所得，P3值为17-AAG+负压处理组与单纯17-AAG组各时间点比较所得，P4值为17-AAG+负压处理组与单纯负压处理组各时间点比较所得；“—”表示无此项 
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表2 4组人脐静脉血管内皮细胞处理完成后培养6 h成管情况比较（
组别 样本数 成管总长度（μm） 分支节点数（个） 正常对照组 3 14 310±1 750 360±55 单纯17-AAG组 3 10 680±1 240 258±46 单纯负压处理组 3 20 670±1 640 660±66 17-AAG+负压处理组 3 13 310±1 560 383±27 F值 22.07 44.63 P值 <0.001 <0.001 P1值 0.022 0.040 P2值 <0.001 <0.001 P3值 0.073 0.017 P4值 <0.001 <0.001 注：F值、P值为4组间各指标总体比较所得；P1值为单纯17-丙烯胺基-17-去甲氨基格尔德霉素（17-AAG）组与正常对照组各指标比较所得，P2值为单纯负压处理组与正常对照组各指标比较所得，P3值为17-AAG+负压处理组与单纯17-AAG组各指标比较所得，P4值为17-AAG+负压处理组与单纯负压处理组各指标比较所得
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表3 3组人脐静脉血管内皮细胞处理完成后细胞中eNOS磷酸化位点1177/eNOS比值与血管新生相关蛋白相对表达量比较（
组别 样本数 eNOS磷酸化位点1177/eNOS比值 eNOS HSP90 窖蛋白1 正常对照组 3 0.531±0.032 1.03±0.11 0.362±0.051 1.08±0.09 单纯负压处理组 3 0.882±0.022 1.03±0.05 0.721±0.043 1.10±0.08 17-AAG+负压处理组 3 0.501±0.041 1.01±0.03 0.503±0.022 1.06±0.10 F值 113.00 0.08 64.21 0.11 P值 <0.001 0.920 <0.001 0.850 P1值 <0.001 — <0.001 — P2值 <0.001 — <0.001 — 注：F值、P值为3组间各指标总体比较所得；eNOS为内皮型一氧化氮合酶，HSP90为热休克蛋白90；P1值为单纯负压处理组与正常对照组各指标比较所得，P2值为17-丙烯胺基-17-去甲氨基格尔德霉素（17-AAG）+负压处理组与单纯负压处理组各指标比较所得；“—”表示无此项
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表4 3组人脐静脉血管内皮细胞处理完成后采用免疫共沉淀与蛋白质印迹法检测细胞中窖蛋白1及eNOS蛋白相对表达量比较（
组别 样本数 窖蛋白1 eNOS 正常对照组 3 1.00±0.10 1.00±0.12 单纯负压处理组 3 1.51±0.08 0.58±0.10 17-AAG+负压处理组 3 0.48±0.05 2.02±0.38 F值 127.12 29.16 P值 <0.001 <0.001 P1值 0.002 0.012 P2值 <0.001 <0.001 注：eNOS为内皮型一氧化氮合酶；F值、P值为3组间各指标总体比较所得；P1值为单纯负压处理组与正常对照组各指标比较所得，P2值为17-丙烯胺基-17-去甲氨基格尔德霉素（17-AAG）+负压处理组与单纯负压处理组各指标比较所得
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脱细胞真皮基质（ADM） 重症监护病房（ICU） 动脉血氧分压（PaO2） 丙氨酸转氨酶（ALT） 白细胞介素（IL） 磷酸盐缓冲液（PBS） 急性呼吸窘迫综合征（ARDS） 角质形成细胞（KC） 反转录-聚合酶链反应（RT-PCR） 天冬氨酸转氨酶（AST） 半数致死烧伤面积（LA50） 全身炎症反应综合征（SIRS） 集落形成单位（CFU） 内毒素/脂多糖（LPS） 超氧化物歧化酶（SOD） 细胞外基质（ECM） 丝裂原活化蛋白激酶（MAPK） 动脉血氧饱和度（SaO2） 表皮生长因子（EGF） 最低抑菌浓度（MIC） 体表总面积（TBSA） 酶联免疫吸附测定（ELISA） 多器官功能障碍综合征（MODS） 转化生长因子（TGF） 成纤维细胞（Fb） 多器官功能衰竭（MOF） 辅助性T淋巴细胞（Th） 成纤维细胞生长因子（FGF） 一氧化氮合酶（NOS） 肿瘤坏死因子（TNF） 3-磷酸甘油醛脱氢酶（GAPDH） 负压伤口疗法（NPWT） 血管内皮生长因子（VEGF） 苏木精-伊红（HE） 动脉血二氧化碳分压（PaCO2） 负压封闭引流（VSD）
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