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作 者:帅维正 刘剑飞[3] 吴成林 刘妍[3] 蒲倩 王欲晓 周丽君[3] 张志成[2] 段蕴铀[1] SHUAI Wei-zheng;LIU Jian-fei;WU Cheng-lin;LIU Yan;PU Qian;WANG Yu-xiao;ZHOU Li-jun;ZHANG Zhi-cheng;DUAN Yun-you(Navy Clinical Medical College, Second Military Medical University, Shanghai 200433, China;Department of ICU, 3Center for Basic Medical Science, the Navy General Hospital, Beijing 100048, China)
机构地区:[1]上海第二军医大学海军临床医学院,上海200433 [2]海军总医院ICU,北京100048 [3]海军总医院中心实验科,北京100048
出 处:《解放军医学杂志》2018年第5期419-423,共5页Medical Journal of Chinese People's Liberation Army
基 金:军队医药卫生重点课题(BHJ14J004)
摘 要:目的探讨创伤弧菌溶细胞素(VVc)对永生化小鼠骨髓来源巨噬细胞(i BMDM)坏死性凋亡的作用及机制。方法 i BMDM细胞按照不同浓度的VVc刺激以及是否接受受体相互作用蛋白1(RIP1)特异性抑制剂Nec-1的预处理分为5组:对照组、VVc 2μg/ml组、VVc 4μg/ml组、Nec-1+VVc 2μg/ml组和Nec-1+VVc 4μg/ml组。Nec-1各组在VVc处理前1h加入5μmol/L Nec-1,实验中各组在VVc处理2h后收集培养上清及细胞。采用乳酸脱氢酶(LDH)法评价VVc对细胞的毒性情况;采用流式细胞仪检测细胞状态;采用Western blotting检测细胞焦亡和坏死性凋亡相关蛋白GSDMD(gasdermin D protein)和混合系列蛋白激酶样结构域(MLKL)的表达。结果培养基上清内LDH水平随VVc浓度上升而升高,且可被Nec-1预处理所抑制(P<0.05)。流式细胞检测结果显示,VVc处理后呈Annexin V/PI双标阳性的细胞比例明显增加,使用Nec-1预处理后,该比例明显下降(P<0.05)。Western blotting检测结果显示,VVc处理组p MLKL水平明显高于对照组和Nec-1+VVc处理组(P<0.05),各组均未观察到焦亡相关蛋白存在剪切体活性形式。结论 VVc可通过RIP1诱导小鼠骨髓来源巨噬细胞发生坏死性凋亡。Objective To investigate the effects of necroptosis induced by vibrio vulnificus cytolysin(VVc) on immortalized bone marrow-derived macrophage cell line(i BMDM) and its mechanism. Methods The i BMDM were divided into five groups, control group, VVc(2μg/ml) group, VVc(4μg/ml) group, Necrostatin-1(Nec-1)+VVc(2μg/ml) group, Nec-1+VVc(4μg/ml) group based on the concentration of VVc and whether the cells received receptor interacting protein 1(RIP1). In the RIP1 inhibited groups, i BMDM was pretreated with Nec-1(5μmol/L) before stimulation with VVc. Cells and culture supernatant were harvested at 2 h after the challenge by cytolysin. Lactate dehydrogenase(LDH) activity in cell-culture supernatant following VVc stimulation was determined to evaluate cell death. The cell state was detected by flow cytometry with Annexin V/propidium iodide staining, and the expression of phosphorylated mixed lineage kinase domain-like(MLKL) and pyroptosis-and necroptosis-related proteins(caspase-1, caspase-11, gasdermin D) were detected by Western blotting. Results The LDH level in the culture supernatant was increased in the groups stimulated by VVc and could also be inhibited by pretreatment with Nec-1(P〈0.05). Compared with control and RIP1-inhibited groups, the VVc-treatment induced a high amount of the double positive staining cells in the VVc group(P〈0.05). In the RIP1 inhibited groups, Nec-1 pretreatment could significantly decrease the levels of p MLKL(P〈0.05), compared with VVc group. None of the cleaved pyroptosis-related protein was detected by Western blotting. Conclusion Necroptosis induced by RIP1 may play an important role in VVc injury model of i BMDM, furthermore, Nec-1 could reduce the degree of injury after VVc challenge.
关 键 词:创伤弧菌 溶细胞素 坏死性凋亡 依赖受体相互作用蛋白
分 类 号:R378[医药卫生—病原生物学]
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