摘要
目的探讨内皮缝隙连接在介导细胞间通讯及在损伤血管内皮修复中的作用。方法采用植块法培养大鼠主动脉内皮细胞,细胞免疫荧光染色法检测大鼠主动脉内皮细胞缝隙连接蛋白37、40及47的表达,荧光漂白后恢复技术检测缝隙连接介导的细胞间通讯。机械划痕建立内皮损伤模型,每24h摄像一次以定量分析内皮损伤修复速度,并观察缝隙连接特异性阻断剂18α-甘草次酸对内皮损伤后修复的影响。结果缝隙连接蛋白37、40及47在内皮细胞中均有表达。荧光物质能够通过缝隙连接在相邻细胞间进行传递,孤立细胞荧光恢复率显著低于相邻细胞(5.70%±0.63%比82.26%±1.68%,P<0.01);而18α-甘草次酸能够抑制缝隙连接介导的细胞间通讯,18α-甘草次酸干预组荧光恢复率显著低于对照组(53.58%±1.73%比82.26%±1.68%,P<0.05)。内皮划痕宽度在对照组与18α-甘草次酸干预组之间无显著性差异(396.57±25.32μm比370.12±19.40μm,P>0.05),内皮损伤24h后,18α-甘草次酸干预组划痕宽度显著大于对照组(237.38±20.40μm比126.29±21.40μm,P<0.05)。18α-甘草次酸干预组内皮损伤完全愈合时间显著多于对照组(4.2±0.2天比2.6±0.3天,P<0.05)。结论内皮细胞间存在缝隙连接介导的细胞间通讯,而18α-甘草次酸能抑制这种细胞间通讯并减慢内皮损伤修复速度及延长其愈合时间,提示内皮细胞缝隙连接在内皮损伤后修复过程中起重要作用。
Aim To explore the role of endothelial gap junctions in intercellular communication and vascular endothelial wound repair. Methods Rat aortic endothelial cells （RAEC） were cultured by explanted rat aortic wall tissue. Cell immunofluorescence staining was applied to detect the expressions of connexin （Cx） 37, Cx40 and Cx47 in RAEC. Fluorescence redistribution after photobleaching （FRAP） was used to measure the communications between cells via gap junctions. The monolayer of cultured RAEC was scraped by a mechanical method and the endothelial wound healing rate was quantified by an analysis of the photographs taken every 24 h after endothelial cell layers were wounded. Meanwhile, 18α-glyeyrrhefinic acid （ 18α-GA）, a specific blocker of gap junction, was administered to observe its effect on endothelial wound repair. Results Cx37, Cx40 and Cx47 were all expressed in RAEC. Fluorescent dye could only be transferred between conjugated cells, and mean fluorescence recovery rate in isolated cells were significantly lower compared with that in conjugated cells （ 5.70% ± 0.63% vs 82.26% ± 1.68%, P 〈 0.01 ）. Compared with control group, mean fluorescence recovery rate in 18α-GA group were significant lower （53.58%±1.73% vs82.26%±1.68%, P〈0.05）. Therefore, 18α-GA could inhibit dye transfer between conjugated cells. The scrape width of endothelium were similar in two groups at the time of wound. However, at 24 h after endothelial wound, the scrape width in 18α-GA group were significantly bigger than those in control group （ 237.38 ± 20.40 μm vs 126.29 ± 21.40 μm, P 〈 0.05）. The complete recover time of the wound in 18α-GA group were significantly more than those in control group（4.2±0.2 d vs 2.6±0.3 d, P〈 0.05）. Conclusions There are intercellular communications via gap junctions among conjugated RAEC and 18α-GA could inhibit the intercellular communication via gap junctions, slow down endothelial wound repair speed and prolong the complete recover time of endothelial wound. Hence, endothelial gap junctions nfight play a vital role in the process of endothelial wound repair.
出处
《中国动脉硬化杂志》
CAS
CSCD
2007年第10期725-728,共4页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金(30570765)资助
