摘要
目的探讨体外共培养模式下活化的小胶质细胞对人脐静脉内皮细胞(HUVECs)紧密连接的影响。方法原代培养大鼠视网膜小胶质细胞分成4组,分别用不同浓度的脂多糖(LPS)(0 ng/mL、10 ng/mL、100 ng/mL和1 000 ng/mL)激活小胶质细胞24 h,通过ELISA检测LPS激活后小胶质细胞分泌肿瘤坏死因子-α(TNF-α)的水平。小胶质细胞接种于Transwell的上室,用含100 ng/mL LPS的DMEM/F12培养24 h后用于实验。共培养体系作如下分组:A组,上室空白,下室HUVECs;B组,上室为未激活的小胶质细胞,下室HUVECs;C组,上室为用100 ng/mL LPS激活24 h的小胶质细胞,下室HUVECs。三组培养体系共培养24 h后,将HUVECs行细胞免疫荧光染色,观察claudin 1的表达,并行Western Blot检测claudin 1和occludin蛋白表达变化。结果四组不同浓度的LPS (0 ng/mL、10 ng/mL、100 ng/mL和1 000 ng/mL)激活小胶质细胞24 h后,小胶质细胞分泌TNF-α的水平不同,分别为(16.36±3.90) pg/mL、(378.46±11.46) pg/mL、(507.11±11.20) pg/mL、(754.55±53.43) pg/mL,组间比较差异有统计学意义(P<0.05);小胶质细胞和HUVECs共培养24 h后,细胞免疫荧光染色显示C组HUVECs的claudin 1的表达较A组和B组减弱;Western Blot检测共培养体系A组、B组和C组的claudin 1和occludin相对蛋白量,claudin 1蛋白相对表达量在A组、B组和C组分别为0.233±0.010、0.244±0.010、0.171±0.001,occludin蛋白相对表达量在A组、B组和C组分别为0.474±0.045、0.323±0.029、0.139±0.017,三组间比较,claudin 1和occludin的相对蛋白量差异均有统计学意义(P<0.05),C组的claudin 1和occludin相对蛋白量下调。A组和B组claudin 1的表达差异无统计学意义(P>0.05)。结论活化的小胶质细胞能产生大量的TNF-α,可能通过上调TNF-α的表达使HUVECs紧密连接蛋白的表达下调。
Objective To investigate the effect of microglia on the expression of tight junction in human umbilical vein endothelial cells(HUVECs) by co-culture in vitro. Methods Primary microglia of rat was cultured and divided into four groups activated with lipopolysaccharide(LPS) in different concentration for 24 h, including normal control group(0 ng/mL LPS), 10 ng/mL LPS group, 100 ng/mL LPS group, and 1 000 ng/mL LPS group. The protein levels of tumor necrosis factor-alpha(TNF-α) in the culture media were detected by enzyme linked immunosorbent assay(ELISA). Retinal microglia were cultured onto Transwell permeable support membrane inserts, then activated by lipopolysaccharide(LPS)(100 ng/mL) for 24 h. HUVECs were randomly divided into three groups: HUVECs with empty inserts without cultured microglia(group A), HUVECs with untreated microglia(group B), and HUVECs with 100 ng/m L LPS-treated microglia(group C). After co-cultured for 24 h, immunocytochemistry was applied to observe claudin1 expression in these HUVECs. The protein expression of claudin 1 and occludin were analyzed by Western blot.Results The protein levels of TNF-α 24 h after activation in the microglia culture media of the four groups(0 ng/mL,10 ng/mL, 100 ng/mL, 1 000 ng/mL LPS) were(16.36±3.90) pg/mL,(378.46±11.46) pg/mL,(507.11±11.20) pg/mL, and(754.55±53.43) pg/mL, with statistically significant differences among the four groups(P<0.05). HUVECs co-cultured with 100 ng/mL LPS-treated microglia(group C), compared to group A and group B, expressed lower level of claudin 1.Claudin 1 protein levels in group A, group B, and group C were 0.233±0.010, 0.244±0.010, 0.171±0.001, respectively.Moreover, the expression of occludin in group A, group B, and group C were 0.474±0.045, 0.323±0.029, 0.139±0.017.Western Blot analysis revealed downregulations of claudin 1 and occludin in HUVECs co-cultured with LPS-activated microglia(P<0.05). Comparison between group A and group B showed that there was no significant differences in the expression of claudin
作者
廖宇洁
于晓彦
金轶平
朱皓皓
LIAO Yu-jie;YU Xiao-yan;JIN Yi-ping;ZHU Hao-hao(Department of Ophthalmology, the Fifth People' s Hospital of Shanghai, Fudan University, Shanghai 200240, CHINA)
出处
《海南医学》
CAS
2019年第5期548-551,共4页
Hainan Medical Journal
基金
上海市闵行区自然科学研究课题(编号:2011MHZ37)
