nlrp3炎症小体参与hsv-1诱导病毒性心肌炎的实验 - 南京医科大学.docVIP

NLRP3炎症小体参与HSV-1诱导病毒性心肌炎的实验研究 宋 宁,陈相健,王子盾,王秀芝,徐东杰* （南京医科大学第一附属医院心血管内科,江苏 南京 210029） [摘 要] 目的:观察NOD样受体热蛋白结构域相关蛋白3(Nod-like receptor pyrin domain-containing protein 3, NLRP3)炎症小体是否参与单纯疱疹病毒1型(Herpes simplex virus-1, HSV-1)诱导的病毒性心肌炎(viral myocarditis, VMC)的病理过程。方法:培养乳鼠心室肌细胞(Neonatal rat ventricular myocytes, NRVM)，分别以0.01PFU和0.1PFU HSV-1感染NRVM，24h后，通过光学显微镜观察细胞形态学改变,实时定量PCR(Quantitative real-time PCR, qRT-PCR)测定NLRP3炎症小体及其下游通路的mRNA表达水平，免疫荧光(immunofluorescence, IF)显示半胱天冬酶-1(cysteinyl aspartate-specific proteases-1，Caspase-1)的表达,全自动生化仪测定细胞上清肌酸激酶同工酶MB(Creatine kinase-MB, CK-MB)的含量以及酶联免疫吸附试验(Enzyme-linked immunosorbent assay, ELISA)测定细胞上清白细胞介素-18(Interleukin-18，IL-18)的浓度。结果：HSV-1感染心肌细胞模型组出现细胞病变现象(Cytopathic effect, CPE)，培养细胞上清细胞损伤标志物CK-MB明显增高(P0.05)，qRT-PCR测定模型组NLRP3、Caspase-1、IL-1β和IL-18 mRNA相对表达量较对照组高5倍以上，IF显示Caspase-1在HSV-1干预后的NRVM胞质内表达明显增高，培养细胞上清IL-18浓度较对照组增高(P0.05)。结论：NLRP3炎症小体及下游通路在HSV-1诱导的VMC细胞模型中被激活，参与其病理过程，为治疗病毒性心肌炎提供可能的新靶点。 [关键词] NLRP3炎症小体;单纯疱疹病毒1型;病毒性心肌炎; 乳鼠心室肌细胞 [中图分类号] R542.2+1 [文献标志码] A Experimental research on NLRP3-inflammasome participated in HSV-1 induced viral myocarditis Song Ning,Chen Xiangjian,Wang Zidun,Wang Xiuzhi,Xu Dongjie* (Department of Cardiology,the First Affiliated Hospital of NJMU, Nanjing 210029,China) [Abstract] Objective: To observe whether the Nod-like receptor pyrin domain-containing protein 3（NLRP3)-inflammasome could participate in the pathologic process of Herpes simplex virus-1（HSV-1）induced viral myocarditis(VMC). Method: Cultured neonatal rat ventricular cardiomyocytes (NRVM) were infected with 0.01 or 0.1 PFU HSV-1 for 24 hours. Morphologic changes of NRVM were observed under light microscope. The gene expression of NLRP3-inflammasome and its downstream pathways were measured by quantitative real-time PCR (qRT-PCR). The expression and location of Caspase-1(cysteinyl aspartate-specific proteases-1) were evaluated by immunofluorescent (IF) method. Moreover, the supernatant concentrations of Interleukin-18(IL-18) and Creatine kin