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目的研究高迁移率族蛋白B1(HMGB1)对内皮细胞通透性的影响,以及HMGB1受体(RAGE)和p38 MAPK通路在此病理过程中的作用。方法 HMGB1(200 ng/m L)与人脐静脉内皮细胞株分别在体外共同培养0、3、6、12和24 h,以0 h为对照检测HMGB1诱导单层内皮细胞通透性Pd改变的时间效应;用电阻法分别在0、3、6、12和24 h测量正常空白对照组(单纯培养基)和HMGB1组(200 ng/m L)的单层内皮细胞电阻值TER,比较两组间在相同时间点电阻值的差别;p38 MAPK抑制剂SB203580(25μmol/L)预先作用1 h,继以HMGB1(200 ng/m L)作用12h,比较不同组间细胞通透性的变化;以HMGB1(200 ng/m L)分别作用0、10、20、30、60 min后观察细胞内p38蛋白表达及磷酸化p38表达的时间效应;转染siRNA下调RAGE表达,或以原代培养野生型和RAGE(-/-)小鼠肺微血管内皮细胞培并继以HMGB1刺激,比较各组细胞内磷酸化p38蛋白表达的变化。通透性检测采用FITC荧光标记右旋糖酐漏出法及跨内皮电阻(TER)测定法,蛋白表达用免疫印迹法检测。结果 HMGB1以时间依赖的方式引起单层内皮细胞通透性Pd的升高,12 h与0 h相比,差异有统计学意义(P<0.05);且可使跨细胞电阻TER降低,在12 h与空白对照组相应时间点相比,差异有统计学意义(P<0.05);也可引发p38蛋白磷酸化水平的升高,与0 min相比,差异有统计学意义(P<0.05);SB203580预处理再以HMGB1刺激后细胞通透性明显降低,与HMGB1单纯刺激组相比,差异有统计学意义(P<0.05);RAGE siRNA转染后以HMGB1刺激细胞p38磷酸化水平显著下调,与HMGB1组相比,差异有统计学意义(P<0.05);HMGB1刺激野生型小鼠PMVECs可诱导p38磷酸化,而RAGE(-/-)小鼠PMVECs则无此效应(P<0.05)。结论 HMGB1通过结合RAGE来激活p38诱导血管内皮细胞通透性的增高。
Objective To investigate the effects of HMGB1 on endothelial cell permeability and the role of HMGB1 receptor (RAGE) and p38 MAPK pathways in this pathological process. Methods HMGB1 (200 ng / mL) and human umbilical vein endothelial cells were co-cultured in vitro for 0, 3, 6, 12 and 24 h, respectively. Time effect. The resistance of monolayer endothelial cells was measured by resistance method at 0, 3, 6, 12 and 24 h respectively in normal control group (simple medium) and HMGB1 group (200 ng / m L) (P <0.05). The difference of the resistance between the two groups at the same time point; the pretreatment of p38 MAPK inhibitor SB203580 (25μmol / L) for 1 h followed by HMGB1 (200 ng / m L) for 12h, HMGB1 (200 ng / mL) for 0, 10, 20, 30 and 60 min, respectively. The expression of p38 and phosphorylated p38 in transfected cells were detected by RT-PCR. And RAGE (- / -) mice lung microvascular endothelial cells were stimulated with HMGB1 stimulation, compared with each group changes in phosphorylated p38 protein expression. Permeability test was performed by FITC fluorescent dextran leakage method and trans-endothelial resistance (TER) assay. The protein expression was detected by Western blotting. Results HMGB1 induced the increase of Pd permeability in monolayer endothelial cells in a time-dependent manner. Compared with 0 h at 12 h, the difference was statistically significant (P <0.05) (P <0.05). The phosphorylation level of p38 protein was also significantly increased compared with the control group (P <0.05). Compared with the control group at 0, the difference was statistically significant (P <0.05) ; SB203580 pretreatment and HMGB1 stimulation significantly decreased cell permeability, compared with HMGB1 stimulation group, the difference was statistically significant (P <0.05); RAGE siRNA transfected HMGB1 stimulated cells p38 phosphorylation level was significantly down-regulated (P <0.05). Compared with HMGB1 group, the difference was statistically significant (P <0.05). PMVECs induced by HMGB1 could induce p38 phosphorylation in PMVECs but no effect in PMVECs (P <0.05) . Conclusion HMGB1 activates p38-induced increase of vascular endothelial cell permeability by binding to RAGE.