生长停滞特异性蛋白6在牙龈卟啉单胞菌脂多糖诱导内皮细胞黏附因子及趋化因子表达中的作用

Role of vitamin K-dependent protein Gas6 in the expression of endothelial cell adhesion molecule-1 and chemokines induced by Porphyromonas gingivalis lipopolysaccharide

目的:探讨维生素K依赖的生长停滞特异性蛋白6(growth-arrest-specific protein 6,Gas6,一种维生素K依赖性蛋白,参与细胞增殖、存活、黏附和迁移,也在炎症反应中起重要作用)在牙龈卟琳单胞菌脂多糖(Porphyromonas gingivalis lipopolysaccharide,P.g-LPS)诱导血管内皮细胞(human umbilical vein endothelial cells,HUVECs)表达黏附因子和趋化因子过程中的作用。方法:在血管内皮细胞中过表达和敲低Gas6基因后,用1 mg/L P.g-LPS刺激血管内皮细胞3 h和24 h,利用实时定量荧光聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)检测细胞间黏附分子1(intercellular adhesion molecule-1,ICAM-1)和E选择素(E-selectin),以及趋化因子白细胞介素-8(interleukin-8,IL-8)和单核细胞趋化蛋白1(monocyte chemoattractant protein 1,MCP-1)的表达。过表达或敲低Gas6基因后,细胞划痕实验检验内皮细胞迁移的生物学功能表型的变化。结果:P.g-LPS刺激3 h后,敲低Gas6组的黏附因子及趋化因子的表达情况与对照组相比,差异无统计学意义(P>0.05),过表达Gas6组的黏附因子及趋化因子与对照组相比显著降低(E-selectin、ICAM-1、IL-8和MCP-1分别下降81%±0%、47%±3%、76%±3%和26%±6%,P<0.01);P.g-LPS刺激24 h后,敲低组的黏附因子及趋化因子表达情况与对照组相比显著升高(E-selectin、ICAM-1、IL-8和MCP-1上调的倍数分别是2.06±0.07、1.99±0.11、3.14±0.15和1.84±0.03),过表达组的黏附因子及趋化因子与对照组相比显著降低(E-selectin、ICAM-1、IL-8和MCP-1分别下降29%±1%、62%±3%、69%±1%和41%±2%),实验组与对照组黏附分子及趋化分子表达差异具有统计学意义(P<0.01)。细胞划痕实验结果显示,敲低Gas6组细胞迁移能力与对照组相比增强,过表达组细胞迁移能力较对照组弱,与realtime PCR检测结果趋势一致。结论:下调Gas6基因后,P.g-LPS促进ICAM-1、E-selectin、IL-8和MCP-1表达作用增强,上调Gas6基因后,P.g-LPS促进ICAM-1、E-selectin、IL-8和MCP-1表达作用减弱,各细胞因子表达量与Gas6表达趋势相反,提示Gas6在内皮细胞炎症状态下的黏附过程中可能发挥抑制作用。

Objective ： Growth-arrest-specific protein 6 （ Gas6 ） is a vitamin K-dependent protein and involved in cell proliferation, survival, adhesion and migration . Also it has been shown to play an impor-tant role in the inflammatory response . The aim of present study was to investigate the role of Gas6 in the process of the expression of adhesion molecules and chemokines of human umbilical vein endothelial cells （HUVECs） induced by Porphyromonas gingivalis lipopolysaccharide （ P. ^ -LPS）. Methods： After up- regulation and down-regulation of the expression of Gas6 , the vascular endothelial cells were stimulated with 1 mg/L P. ^-LPS for 3 h and 24 h. Real-time quantitative polymerase chain reaction （ real-time PCR）was taken to detect the expression of the cell adhesion molecules ： intercellular adhesion molecule-1 （ICAM-1） and E-selectin, as well as chemokines ： interleukin-8 （ IL-8 ） and monocyte chemoattractant protein 1 （ MCP-1） . Wound healing assay was taken to observe the migration ability of endothelium cellsin different groups. Results ： After 3 h of P. g - LPS stimulation, the expression of adhesion molecules and chemokine in the down-regulation group was not significantly different from that in the control group,while in the up-regulation group the decrease of E- selectin, ICAM-1,IL-8 and MCP-1 was 8 1 % ± 0 % , 47% ±3%,76% ± 3%,26% ± 6% respectively. After 24 h of R LPS stimulation,the expression of adhesion molecules and chemokine in down-regulation group was significantly higher than that in con-trol group （2. 06 ±0. 07,1.99 ±0.11,3.14±0. 15,1.84 ±0. 03 flod）,while these molecules in the down-regulation group was significantly lower than in the control group （29% ±1%,6 2 % ± 3 %, 69% ±1%,41% ±2%）. Differences were statistically significant （P 〈0. 01 ）.Wounding healing assay showed that down-regulation of Gas6 enhanced migration ability of endothelial cells while up-regulation of Gas6 weakened this ability, which was consistent with the trend of real-time PCR result. Conclusion ： Down-regulation of the Gas6 gene enhanced the expression of ICAM-1, E- selectin,IL-8 and MCP-1 in HUVECs after P. g - LPS stimulating, while up-regulaiton of the Gas6 gene weakened the expression of ICAM-1, E- selectin, IL-8 and MCP-1 in HUVECs after P. g - LPS stimulating, suggesting that Gas6 may play a role in the process of endothelial cell adhesion.