核苷酸结合寡聚结构域受体1作为先天免疫识别受体在血管平滑肌细胞激活中的作用

Role of nucleotide binding oligomerization domain receptor 1 as a new innate immune recognition receptor in activation of vascular smooth muscle cells

目的:核苷酸结合的寡聚结构域受体1是新近发现的先天免疫细胞内模式识别受体。实验通过观察细菌细胞壁粘肽对血管平滑肌中细胞内核苷酸结合的寡聚结构域受体1激活和表达的影响,探讨核苷酸结合的寡聚结构域受体1介导的先天免疫信号通路在血管平滑肌激活中的作用。方法:实验于2006-06/2007-03在大连医科大学附属二院中心实验室完成。①实验材料:人冠状动脉血管平滑肌细胞购自Cambrex公司。②实验过程:体外培养人冠状动脉血管平滑肌细胞,用核苷酸结合的寡聚结构域受体1激动剂细菌细胞壁粘肽(10mg/L)刺激0,3,6和24h。③实验评估:用实时定量反转录-聚合酶链反应检测平滑肌细胞中核苷酸结合的寡聚结构域受体1mRNA的表达,用酶联免疫吸附法检测细胞培养物上清液中白细胞介素8和肿瘤坏死因子α的浓度。结果:①血管平滑肌细胞在静息状态下表达低水平的核苷酸结合的寡聚结构域受体1。细菌细胞壁粘肽刺激能够引起血管平滑肌细胞中核苷酸结合的寡聚结构域受体1mRNA表达上调(从0.164±0.005增加到0.231±0.027,P<0.05)。②酶联免疫吸附法显示细胞培养物上清液中白细胞介素8和肿瘤坏死因子α的浓度也显著增加[白细胞介素8从(0.12±0.01)μg/L增加到(2.31±0.11)μg/L,肿瘤坏死因子α从(4.22±0.50)ng/L增加到(143.11±12.58)ng/L,P<0.01]。结论:推测血管平滑肌细胞中的核苷酸结合的寡聚结构域受体1介导的先天免疫信号通路被激活,参与了动脉粥样硬化的发生和发展。

AIM： Nucleotide binding oligomerization domain receptor 1 （NOD1） is a recently identified intracellular pathogen pattern recognition receptor of innate immunity. This study was designed to explore the role of NODl-mediated innate immune signal pathway in the activation of vascular smooth muscle cells （VSMCs）, and investigate the effect of peptidoglycan （PGN） on the activation and expression of NOD1 in human VSMCs. METHODS： The experiment was performed in the central laboratory of the Second Affiliated Hospital of Dalian Medical University from June 2006 to March 2007.①Subject： Human coronary artery VSMCs were purchased from Cambrex company.② Methods： Human coronary artery VSMCs were cultured in vitro, and stimulated with NOD1 agonist PGN （10 mg/L） for 0, 3, 6 and 24 hours. ③ Evaluation： The mRNA expression of NOD1 in VSMCs was measured by real time quantitative reverse transcription-polymerase chain reaction. The concentration in the culture supernatants of interleukin-8 （IL-8） and tumor necrosis factor-α （TNF-α ） was determined by enzyme linked immunosorbent assay. RESULTS： ①Human VSMCs constitutively expressed a low level of NOD1 at resting condition. Upon PGN stimulation, the expression of NOD1 mRNA was up-regulated in VSMCs, from 0.164±0.005 to 0.231 ±0.027 （P 〈 0.05）.②Enzyme linked immunosorbent assay showed the concentration in the culture supernatants of IL-8 and TNF-α increased significantly, IL-8 from （0.12±0.01） μg/L to （2.31 ±0.11） μg/L and TNF-α from （4.22±0.50） ng/L to （143.11 ± 12.58） ng/L, respectively （P 〈 0.01）. CONCLUSION： The NODl-mediated innate immune pathway may be involved in the atherogenesis though the activation of VSMCs.