过表达pellino-1在Kupffer细胞内毒素耐受时对TRAF3泛素化及MAPK信号通路的调控

发布时间：2018-03-07 12:38

  本文选题：kupffer细胞　切入点：内毒素耐受　出处：《中山大学学报(医学科学版)》2017年05期 　论文类型：期刊论文

【摘要】：【目的】探讨上调pellino-1在Kupffer细胞(KC)内毒素耐受时对肿瘤坏死因子受体相关因子3(TRAF3)泛素化、促分裂原活化蛋白激酶(MAPK)信号通路及下游细胞因子分泌情况的影响。【方法】分离、培养C57BL/6小鼠KC,随机分为2组:(1)空载对照组:转染空载质粒48 h后,先给予小剂量LPS(10 ng/mL)刺激24 h,再给予大剂量LPS(300 ng/mL)刺激。(2)过表达组:过表达pellino-1慢病毒转染48 h后,先给予小剂量LPS(10 ng/mL)刺激24 h,再给予大剂量LPS(300 ng/mL)刺激。两组分别于处理后0、5、10、30、60 min收获细胞,蛋白质免疫印迹试验(Western blot)检测pellino-1、K48泛素化(K48ubiquitin,K48-Ub)、TRAF3、JNK、p-JNK、p38、p-p38蛋白水平表达变化;酶联免疫吸附法(ELISA)检测细胞培养上清液中IL-1β、TNF-α及IL-10的分泌情况。【结果】与空载对照组相比,过表达组pellino-1蛋白表达量在各个时间点均明显升高;K48-Ub水平明显升高;TRAF3蛋白表达量明显降低;JNK、p38蛋白表达量没有明显变化,但p-JNK及p-p38蛋白表达量显著升高;过表达组细胞上清中IL-1β及TNF-α的量明显升高(P0.05),而IL-10的量则明显降低(P0.05)。【结论】过表达pellino-1可促进TRAF3蛋白K-48泛素化降解,导致TRAF3蛋白表达量降低,激活下游的MAPK信号,从而抑制内毒素耐受的形成。
[Abstract]:[objective] to investigate the effect of upregulation of pellino-1 on Ubiquitin, mitogen-activated protein kinase (MAPK) signaling pathway and cytokine secretion in Kupffer cells during endotoxin tolerance. The cultured C57BL / 6 mice were randomly divided into two groups: control group: after 48 h of transfection of empty plasmid, the mice were stimulated with small dose of LPS(10 ng / mL for 24 h, and then stimulated with large dose of LPS(300 ng / mL. (2) overexpression of pellino-1: 48 h after transfection of pellino-1 lentivirus. Small dose of LPS(10 ng / mL was given for 24 h, and then high dose of LPS(300 ng / mL). The expression of pellino-1k48 ubiquitin K48-UbC3TRAF3KNKp-JNKP p38 p38 protein was detected by Western blotting in the two groups at 0 ~ 5 ~ 10 ~ 10 ~ 30 ~ 60 min ~ (-1) ~ (th) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (?). Enzyme linked immunosorbent assay (Elisa) was used to detect the secretion of IL-1 尾 -TNF- 伪 and IL-10 in the supernatant of cell culture. In the overexpression group, the expression of pellino-1 protein increased significantly at each time point, the level of K48-Ub increased significantly, the expression of TRAF3 protein decreased significantly, but the expression of p-JNK and p-p38 protein increased significantly. In overexpression group, the amount of IL-1 尾 and TNF- 伪 in supernatant increased significantly, while the amount of IL-10 decreased significantly. [conclusion] overexpression of pellino-1 can promote the degradation of K-48 ubiquitin of TRAF3 protein, decrease the expression of TRAF3 protein and activate the downstream MAPK signal. Thus inhibiting the formation of endotoxin tolerance.
【作者单位】： 重庆医科大学附属第二医院肝胆外科;重庆医科大学附属第一医院肝胆外科;
【基金】：国家自然科学青年基金(81401622;81301656) 重庆市科委基础科学与前沿技术研究专项(重点,cstc2015 jcyjBX0070)
【分类号】：R459.7
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