脂多糖与高迁移率族蛋白B1联用对HepG2细胞释放细胞因子的影响  

Combined effect of high mobile group box-1 protein and lipopolysaccharide on release of cytokines from human HepG2 cells

作　　者：刘志锋[1] 王娟[2] 刘铮[2] 唐柚青[1] 孟繁甦[1] 刘靖华[2] 苏磊[2] 

机构地区：[1]广州军区广州总医院ICU,广东510010 [2]南方医科大学省部共建功能蛋白质组学重点实验室,广东广州510515

出　　处：《中国危重病急救医学》2009年第9期532-535,共4页Chinese Critical Care Medicine

基　　金：中国博士后科学基金项目（20080431419）

摘　　要：目的观察脂多糖（LPS）与高迁移率族蛋白B1（HMGB1）单用或联用对人肝癌细胞释放细胞因子的诱导作用。方法培养人肝癌细胞株HepG2，用原核重组表达载体pET14b—HMGB1原核表达纯化HMGB1蛋白；用不同浓度的HMGB1蛋白（0、0．01、0．1、1、10mg／L）或不同浓度的LPS（0、0．1、1、10、100mg／L）以及1mg／LHMGB1和10mg／LLPS联用分别刺激培养的HepG2，24h后收集细胞上清液，用LiquiChip液相蛋白芯片系统检测上清液中粒-巨噬细胞集落刺激因子（GM—CSF）、γ-干扰素（IFN-γ）、白细胞介素-1β（IL-1β）、IL-2、IL-4、IL-6、IL8、IL-10、IL-12、肿瘤坏死因子-α（TNF—α）10种细胞因子的表达水平。结果LPS可以剂量依赖性的方式刺激HepG2分泌IL-6、IL-8表达（P〈0．05或P〈0．01），而对GM—CSF、IFN-γ、IL—1β、IL-2、IL-4、IL-10、IL-12、TNF—α等作用不明显；不同浓度的HMGB1刺激HepG2后发现，低浓度的HMGB1可明显上调IL-6、IL-8的表达（P均〈0．01），但随着浓度升高，这种诱导作用逐渐减弱，并逐渐恢复至对照水平，而对其他8种细胞因子也无明显诱导作用。HMGB1和LPS联用时，高浓度的HMGB1可明显抑制LPS对HepG2分泌IL-6、IL-8的诱导作用（P均〈0．01）。结论人肝癌细胞株HepG2对炎性刺激仅能产生很少种类的细胞因子，其中LPS作用可以上调IL-6和1L-8水平，而HMGB1则表现出量效的反作用，并且高浓度的HMGB1可明显抑制LPS对HepG2分泌细胞因子的诱导作用。Objective To study the effects of high mobile group box-1 protein （HMGB1） and lipopolysaccharide （LPS） singly or in combination on release of cytokines from human liver carcinoma cell line （HepG2）. Methods HepG2 cells were cultured, and purified HMGB1 protein was prepared by chromatography on Ni2--NTA Sepharose column under natural conditions with recombinant expression plasmid pET14b-HMGB1. Different concentrations of HMGB1 （0, 0.01, 0.1, 1, 10 rag/L） and LPS （0, 0.1, 1, 10, 100 mg/L） were added into the cultured ceils for 24 hours, respectively. Then the supernatant were collected to detect the levels of granulocyte/macrophage colony stimulating factor （GM-CSF）, interferon γ （IFN-γ）, tumor necrosis factor-α（TNF-α）, and interleukin-1β] （IL-1β）, IL-2, IL-4, IL-6, IL-8, IL-10, and IL 12 by using LiquiChip system. Lastly, HepG2 cells were costimulated with 10 mg/L LPS and 1 mg/L HMGB1 for 24 hours. The supernatant were collected to determine the levels of above ten of cytokines. Results The expression and release of IL-6 and IL-8 increased from HepG2 ceils after being stimulated by LPS in a dose-dependent manner, but there were no changes in other eight kinds of cytokines （P〈0.05 or P〈0.01）. Low concentration of HMGB1 could up-regulate the expression of IL-6 and IL-8 in HepG2 cells （both P〈0.01）. But the extent of induction decreased with higher concentration of HMGB1. Similar to LPS, there was no effect of HMGB1 on the expression of other eight kinds of cytokines from cultured HepG2 cells. Furthermore, high concentration of HMGB1 could obviously inhibit the upregulation of IL-6 and IL-8 by high concentration of LPS when the HepG2 cells were costimulated with LPS and HMGB1 （both P〈0.01）. Conclusion HepG2 cells could only express and release a few kinds of cytokines when the cells were stimulated with pro-inflammatory agents, such as LPS or HMGB1. Two kinds of cytokines, IL-6 and IL-8 could be up-regulated by LPS and low concentration of HMGB1, and HMGB1

关 键 词：脂多糖 高迁移率族蛋白B1 HEPG2细胞 细胞因子 脓毒症 

分 类 号：R686[医药卫生—骨科学]