安徽医科大学学报

目的 探究CD151通过调控囊泡内化再循环对血管通透性的影响及机制。方法 野生型小鼠和CD151敲除小鼠分别分为WT-con组、WT-模型组、KO-con组和KO-模型组，每组6只。WT-模型组和KO-模型组采用腹腔注射脂多糖(LPS)制备脓毒症急性肺损伤(ALI)模型，WT-con组和KO-con组腹腔注射磷酸盐缓冲液(PBS)作为对照。造模后24 h,通过Miles实验测定肺血管通透性；将沉默CD151表达的siRNA(si-CD151)和阴性对照si-NC转入EA.hy 926细胞，分别观察基础条件及血管内皮生长因子(VEGF-A)刺激条件下，内皮细胞层在不同时间点对FITC-dextran的通透性；CD151敲低组和对照组内皮细胞行转录组测序；免疫荧光检测各组细胞CD151分布及内化；Western bolt及RT-qPCR检测CD151敲低组及对照组VE-cadherin表达情况，免疫荧光检测各组细胞VE-cadherin分布及内化。结果 Miles实验结果提示，WT-模型组小鼠肺组织中染料渗出相较WT-con组增加(P<0.01),KO-模型组小鼠肺组织中染料渗出相较WT-模型组增加(P<0.05)。内皮细胞层通透性检测结果提示，在VEGF-A刺激30、60和120 min后，CD151敲低组对FITC-dextran的通透性显著高于对照组(P<0.05)。转录组测序结果提示，内皮细胞中CD151与囊泡介导的转运存在密切关联。Western bolt和RT-qPCR结果提示，CD151敲低的内皮细胞中VE-cadherin在蛋白和mRNA水平表达较对照组显著下降(均P<0.01)。免疫荧光染色结果提示，在VEGF-A刺激后，与对照组比较，CD151表达下降显著破坏细胞-细胞连接处VE-cadherin的表达，降低了CD151和VE-cadherin在核周区域的共定位。结论 CD151的缺失影响内皮细胞囊泡内化再循环，影响VE-cadherin的表达与内化活动，进而影响血管通透性。

Objective To explore the effect and mechanism of CD151 on vascular permeability by regulating vesicle internalization and recycling. Methods Wild-type mice and CD151 knockout mice were divided into WT-con group, WT-model group, KO-con group and KO-model group, with 6 mice in each group. WT-model group and KO-model group were intraperitoneally injected with LPS to prepare sepsis ALI model, and WT-con group and KO-con group were intraperitoneally injected with phosphate buffer saline(PBS) as a control. 24 h after modeling, pulmonary vascular permeability was measured by Miles test. The siRNA silencing CD151 expression(si-CD151) and negative control si-NC were transfected into EA.hy 926 cells. The permeability of endothelial cell layer to FITC-dextran at different time points was observed under basic conditions and vascular endothelial growth factor-A(VEGF-A) stimulation conditions. Transcriptome sequencing of endothelial cells in si-CD151 group and si-NC group; the distribution and internalization of CD151 in each group were measured using immunofluorescence. Western blot and real-time quantitative RT-qPCR were used to detect the expression of VE-cadherin in si-CD151 groupand other groups. The distribution and internalization of VE-cadherin in each group were measured using immunofluorescence. Results Miles experiment results indicated that dye exudation in lung tissue of WT-model group was significantly higher than that of WT-con group(P<0.01). The dye exudation in the lung tissue of KO-model group increased compared with WT-model group(P<0.05). The results of endothelial cell layer permeability test showed that the permeability of FITC-dextran in si-CD151 group was significantly higher than that in control group after VEGF-A stimulation for 30, 60 and 120 min(P<0.05). Transcriptome sequencing results suggested that CD151 in endothelial cells was closely related to vesicle-mediated transport. Compared with other groups, protein and mRNA levels of VE-cadherin in CD151 knockdown endothelial cells was significantly lower(all P<0.01). The immunofluorescence assay demonstrated that after VEGF-A stimulation, the decrease of CD151 expression significantly impaired the expression of VE-cadherin at cell-cell contacts and reduced the CD151-VE-cadherin colocalization in the perinuclear region compared with other groups. Conclusion The absence of CD151 affects the internalization and recycling of endothelial cell vesicles, affects the expression and internalization of VE-cadherin, and then influences vascular permeability.