摘要
目的研究细胞内模式识别受体核苷酸结合的寡聚结构域(NOD)2受体在血管平滑肌细胞中的表达及其诱导血管平滑肌细胞产生促炎症细胞因子过程中的作用,并探讨它与Toll样受体(TLR)2、4在此过程中的相互关系。方法体外培养人冠状动脉血管平滑肌细胞,用NOD2激动剂胞壁酰二肽(MDP)、TLR2激动剂(PAM3)和TLR4激动剂脂多糖(LPS)单独或联合进行刺激,用实时定量逆转录聚合酶链反应检测血管平滑肌细胞中NOD2和成纤维生长因子2mRNA的表达,用酶联免疫吸附法测定细胞培养物上清液中白介素8和肿瘤坏死因子α的浓度,用四甲基偶氮唑盐微量酶反应比色法检测血管平滑肌细胞增殖活性。结果MDP能使血管平滑肌细胞中NOD2mRNA的表达呈时间依赖性上调[0h:(0.028±0.001);3h:(0.045±0.002);6h:(0.053±0.002);24h:(0.162±0.013)],并能引起血管平滑肌细胞中成纤维生长因子2mRNA的表达增加[MDP组:(9.3±0.4)vs对照组:(7.4±0.2);P<0.05],细胞培养物上清液中白介素8[MDP组:(2.4±0.2)μg/Lvs对照组:(1.0±0.1)μg/L;P<0.05]和肿瘤坏死因子α[MDP组:(51.9±4.7)ng/Lvs对照组:(29.4±3.7)ng/L;P<0.05]的分泌增多和细胞增殖活性增加[(0.90±0.05vs对照组:0.72±0.02;P<0.05]。MDP还能协同LPS、PAM3诱导血管平滑肌细胞增殖活性增加,细胞培养物上清液中白介素8和肿瘤坏死因子α的分泌增加。结论NOD2使血管平滑肌细胞增殖活性增加,诱导其分泌促炎症细胞因子。NOD2与TLR2、4在促进血管平滑肌细胞增殖活性增加,诱导血管平滑肌细胞分泌促炎症细胞因子的过程中具有协同作用。
Objective To investigate the expression of nucleotide-binding oligomerization domain 2 （NOD2）, an intracelluar pathogen pattern recognition receptor, and Toll like receptor （TLR） 2, 4 in human vascular smooth muscle cells （VSMC）, and its effect on production of proinflammatory cytokines in VSMC. Methods Human coronary artery smooth muscle cells were in vitro stimulated with NOD2 agonist Muramyl dipeptide （MDP）, TLR2 agonist Pam3CSK4（PAM3）and TLR4 agonist lipopolysaccharides （LPS） alone or MDP in cocultured with either PAM3 or LPS. The mRNA expression of NOD2 and fibroblast growth factor-2（FGF-2） were measured by real time RT-PCR. The concentration in the culture supernatants of interleukin-8（IL-8） and tumor necrosis factor-α （TNF-α） was determined by ELISA. VSMC proliferation was analyzed by the MTT assay. Results MDP up-regulate the expression of NOD2 mRNA in VSMC in a time-dependent manner （0 h：0. 028± 0.001; 3h： 0. 045±0. 002; 6h： 0. 053±0. 002; 24h： 0. 162±0.013）. It up-regulate the expression of FGF-2 mRNA （MDP 9. 3±0. 4 vs control 7.4±0. 2; P〈0. 05） in VSMC, induce the production of IL-8（MDP 2.4±0. 2 vs control 1.02±0. 13 μg/L） and TNF-α（MDP 51.9±4. 7 vs control 29.4±3.73 ng/L; respectively, P〈0. 05） increases the proliferation of VSMC （MDP 0. 87±0. 05 vs control 0. 72±0.02, P〈0. 05）. Additionally, MDP cocultured with LPS and PAM3 synergetically increase the proliferation of VSMC and the production of IL-8 and TNF-α. Conclusion The activation of NOD2 increase the proliferation of VSMC and the production of proin-flammatory cytokines in VSMC. NOD2 and TLR2 and TLR4 synergetically enhanced these effect.
出处
《中华高血压杂志》
CAS
CSCD
北大核心
2007年第12期1017-1021,共5页
Chinese Journal of Hypertension
