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目的观察普通肝素(UFH)对高迁移率族蛋白1(HMGB 1)介导的人脐静脉内皮细胞屏障通透性损伤的保护作用,探讨UFH对HMGB 1介导的胞质紧密粘连蛋白-1(ZO-1)表达缺失的保护机制。方法将人脐静脉内皮细胞进行体外培养,人脐静脉内皮细胞株传代培养后分为4组(n=5):空白对照组(加入等量PBS)、HMGB 1处理组(100 ng/ml)、UFH对照组(UFH 10 U/ml)、HMGB 1及UFH处理组(100 ng/ml HMGB 1+UFH 10 U/ml)。MTT法测定内皮细胞存活率,用Transwell小室法测定单层内皮屏障通透性,用免疫荧光染色法测定ZO-1的表达分布,蛋白免疫印迹(Western blot)法检测ZO-1蛋白及核因子-κB(NF-κB)的表达。结果 HMGB 1(100 ng/ml)对内皮细胞活性无抑制作用(P>0.05)。UFH预处理后可减少HMGB1所致的内皮细胞通透性增加(P<0.05)。UFH预处理后可降低HMGB 1所致内皮细胞ZO-1密闭环的减少和破坏,使ZO-1荧光强度增强,ZO-1蛋白表达增加,并使NF-κB的核易位减少。结论 UFH可保护HMGB 1介导的内皮细胞ZO-1的表达缺失,进而改善内皮细胞屏障通透性,其机制与减少NF-κB核易位相关。
Objective To investigate the protective effect of unfractionated heparin (UFH) on high mobility group box 1 (HMGB 1) -mediated barrier injury in human umbilical vein endothelial cells (HUVECs), and to investigate the effect of UFH on HMGB 1 -mediated cytoplasmic adhesionin- (ZO-1) loss of expression protection mechanism. Methods Human umbilical vein endothelial cells were cultured in vitro. Human umbilical vein endothelial cells were subcultured and divided into 4 groups (n = 5): blank control group (with equal volume of PBS), HMGB 1 treatment group (100 ng / ml) , UFH control group (UFH 10 U / ml), HMGB 1 and UFH treatment group (100 ng / ml HMGB 1 + UFH 10 U / ml). The survival rate of endothelial cells was determined by MTT method. The permeability of monolayer endothelial barrier was measured by Transwell chamber method. The expression of ZO-1 was detected by immunofluorescence staining. The expression of ZO-1 protein and nuclear factor -κB (NF-κB) expression. Results HMGB 1 (100 ng / ml) had no inhibitory effect on endothelial cell activity (P> 0.05). Pretreatment with UFH decreased the permeability of endothelial cells induced by HMGB1 (P <0.05). Pretreatment with UFH reduced the reduction and destruction of the ZO-1 closed loop of endothelial cells induced by HMGB 1, enhanced the fluorescence intensity of ZO-1, increased the expression of ZO-1 protein and decreased the nuclear translocation of NF-κB. Conclusion UFH can protect the HMGB 1 -mediated expression of ZO-1 in endothelial cells and further improve the barrier permeability of endothelial cells. Its mechanism is related to the reduction of NF-κB nuclear translocation.