铅暴露对体外血脑脊液屏障功能及ZO-1和闭合蛋白表达的影响

Effect of lead exposure on function of blood-cerebrospinal fluid barrier and ZO-1 and occludin protein expression in vitro

目的通过建立体外血脑脊液屏障(BCB)模型,研究铅诱导BCB损伤的可能机制。方法使用Z310细胞建立BCB体外细胞模型,分别加入Pb(AC)22.5,5.0和10.0 mmol·L-1,暴露24,48和72 h。采用跨内皮电阻(TEER)检测法及葡聚糖法检测BCB模型通透性;Western蛋白印迹法和免疫荧光法检测BCB中紧密连接蛋白ZO-1和闭合蛋白表达及分布。结果与细胞对照组相比,Pb(AC)22.5,5.0和10.0 mmol·L-1暴露48 h对Z310细胞存活率和密度均无明显影响。暴露后第12天,与细胞对照组比较,Pb(AC)25.0和10.0 mmol·L-1组TEER值明显降低(P<0.05),葡聚糖通过率显著增高(P<0.05)。Western蛋白印迹法和免疫荧光法检测结果显示,与细胞对照组相比,Pb(AC)2各浓度暴露48 h后,ZO-1和闭合蛋白表达水平明显降低(P<0.05)。结论铅暴露能够破坏BCB模型的通透性,其机制可能与抑制紧密连接蛋白的表达有关。

OBJECTIVE To establish an in vitro blood-cerebrospinal fluid barrier（BCB） model to investigate the underlying mechanism of lead-induced BCB injuries. METHODS The in vitro BCB model was established by Z310 cells. Different concentrations of Pb（AC）2（2.5, 5.0 and 10.0 mmol·L-1） were used for 24, 48 and 72 h. Transendothelial electrical resistance（TEER） and flux of FITC-dextran were performed to determine the permeability of the in vitro BCB model. Western blotting and immunofluorescence methods were used to observe the expression of tight junction protein ZO-1 and occludin. RESULTS Compared with control group, Pb（AC）22.5, 5.0 and 10.0 mmol·L-1exposure for 48 h to Z310 cells had no significant effect on survival rate and density. TEER in different groups was gradually increasing. At the 12 thday after Pb（AC）2exposure, the values of TEER and flux of FITC-dextran in Pb（AC）25 and10 mmol·L-1groups were significantly decreased（P〈0.05）. Western blotting and immunofluorescence images showed that the expression of ZO-1 and occludin were significantly decreased（P〈0.05） after Pb（AC）2exposure for 48 h. CONCLUSION Lead exposure can cause the breakdown of BCB barriers,and this effect may be mediated by reducing the expression of ZO-1 and occludin proteins.