重组表达的猪溶素及其突变体诱导小鼠巨噬细胞的细胞毒性和IL-1β释放  

Suilysin and its mutant induced cytotoxicity and IL-1β release in mouse macrophages

作　　者：任志鸿[1] 黄元铭[1] 王淑京[1] 杜华茂[2] 宋利琼[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室,北京100026 [2]西南大学,重庆404100

出　　处：《疾病监测》2014年第4期276-280,共5页Disease Surveillance

基　　金：传染病预防控制国家重点实验室出国人员留学启动基金(No.2011SKLID301);国家自然科学基金(No.81371761)~~

摘　　要：目的观察猪溶素（Suilysin，SLY）及其无溶血活性的突变体诱导小鼠腹腔巨噬细胞（peritoneal macrophage，PM）释放IL-1β和细胞毒的差异，为进一步研究SLY诱导炎性体激活通路奠定基础。方法重组表达纯化SLY及第353氨基酸位点突变体rSLY^353L，体外和小鼠PM相互作用，用乳酸脱氢酶释放法检测细胞毒作用，用酶联免疫吸附试验（ELISA）检测IL-1β的水平。结果不同浓度rSLY（0．1～10μg）诱导小鼠PM细胞毒性呈现明显的剂量效应，rSLY（1μ/ml）诱导产生750pg／ml的IL-1β同时细胞毒〈20％；rSLY（5μg/ml）可引起最大量的IL-1μ释放但细胞毒高达90％。相同剂量的rSLY^353L不能诱导可检测到的IL—1β和细胞毒性。rSLY与PM共孵育20hIL-1β产量最高，20h后IL—1β产量下降但细胞毒性随孵育时间延长而增大。结论1μ/ml SLY和PM作用20h可诱导较高的IL-1β产生同时细胞毒性较低，该实验条件适用于进一步进行SLY激活PM炎性体信号通路的机制研究。Objective To observe the difference in IL-1β production and cytotoxicity induced by recombinant suilysin （rSLY） and non-hemolytic mutant of SLY, rSLY^353L in mouse peritoneal macrophages （PM） for the better understanding of the mechanism of inflammasome activation induced by SLY. Methods Lactate dehydrogenase （LDH） release assay was conducted to determine the cytotoxicity of rSLY and rSLY^353L. IL-1β production was determined with ELISA. Results The SLY at different concentrations （0. 1 - 10 μg） induced significant cytotoxicity in PM in a dose and time dependent manner, whereas rSLY^353L did not induce detectable cytotoxicity. In contrast to rSLY^353L, 1 μg/ml of rSLY induced 750 pg/ml of IL-lβ and caused less than 20% of cytotoxicity. 5 μg/ml of SLY induced higher level of IL-1β and higher cytotoxicity at the same time. The peak time of IL-1β production by PM induced by rSLY is 20 h after treatment, then the production of IL-1β decreased and the cytotoxicity increased with incubation time. Conclusion SLY （ 1 μg/ml） induced significant IL-1β production and low cytotoxicity by PM at 20 h after treatment which is the optimized condition for further study to understand rSLY-induced inflammasome activation mechanism.

关 键 词：猪溶素 炎症因子 细胞毒性 

分 类 号：S852.611[农业科学—基础兽医学]