摘要
目的通过体外培养SD大鼠大脑皮层微血管内皮细胞并制作划痕损伤模型,观察划痕损伤对脑微血管内皮细胞NF-κB表达及其自噬、凋亡和坏死的影响。方法体外培养SD大鼠大脑皮层微血管内皮细胞并制作划痕损伤模型,应用NF-κB抑制剂吡咯醛二硫氨基甲酸(PDTC)干预划痕损伤脑微血管内皮细胞。随机分为对照组、单纯损伤组、损伤+抑制组,在不同时间点(损伤后1、6、12、24及48 h)分别应用免疫荧光细胞化学法测定磷酸化NF-κB p65蛋白的表达,Annexin V/PI染色法检测凋亡、坏死,Western blot检测自噬蛋白LC3Ⅱ表达情况。对照组为无划痕损伤的脑微血管内皮细胞。结果脑微血管内皮细胞划痕损伤后NF-κB蛋白表达于伤后1 h开始表达增强,于24 h达高峰,与对照组比较差异有统计学意义(P<0.05)。凋亡、坏死及自噬各指标均随时间推移有不同程度升高,与对照组比较差异有统计学意义(P<0.05)。而经PDTC处理后上述变化均受不同程度的抑制,与单纯损伤组相比差异有统计学意义(P<0.05)。结论划痕损伤后可激活脑微血管内皮细胞NF-κB表达,同时可引起脑微血管内皮细胞的自噬、凋亡和坏死。
Objective To culture brain microvascular endothelial cells(BMECs) of SD rats in vitro and construct a model of scratching injury, so as to observe the effect of scratching injury on NF-κB expression as well as auto-phagy, apoptosis and necrosis of of BMECs. Methods BMECs of SD rats were cultured in vitro and scratching injury was implemented, then the cells were treated with pyrrolidine dithiocarbamate(PDTC), an NF-κB inhibitor. The cells were randomly devided into control group, simple-injury group and injury-inhibition group. At 1, 6, 12, 24 and48 hours after injury, NF-κB p65 expression was measured using immunofluorescent cytochemistry assay, apoptosis and necrosis of the cells were determined by Annexin V/PI staining, and expression of autophagy-related protein LC3 II was determined by Western blot. An uninjured group of BMECs was set as control. Results After scratching injury, NF-κB expression of the BMECs was enhanced from 1 h after injury, and peaked at 24 h, significantly higher than that of the control group(P 〈0.05). Indicators for apoptosis, necrosis and autophagy all increased to different extents as time went on, and they showed significant differences from those of the control group(P 〈0.05); while PDTC treatment following injury significantly inhibited these changes(P 〈0.05). Conclusions Scratching damage activates NF-κB expression of BMECs and causes autophagy, apoptosis and necrosis of BMECs.
出处
《中国现代医学杂志》
CAS
北大核心
2016年第5期11-16,共6页
China Journal of Modern Medicine
基金
2012年广东医学院附属医院青年科研基金(No:Qk1308)
2013年广东医学院青年科研基金(No:XQ1315)
2014年度湛江市非资助科技攻关计划项目(No:湛科[2014]27号2014B101)