大肠杆菌DNA诱导全身性炎症反应综合征的作用机制研究

Mechanism of systemic inflammatory response syndrome induced by EC DNA

目的 :探讨细菌DNA是否参与全身性炎症反应综合征 (SIRS)的发生及其可能机制。方法 :碱裂解法抽提纯化大肠杆菌DNA(ECDNA) ;观察ECDNA攻击小鼠的死亡率及大鼠血清TNF -α、IL - 6的变化情况 ;体外培养小鼠单核细胞株ANA - 1细胞 ,采用不同终浓度的ECDNA、LPS刺激ANA - 1细胞 ,测定细胞培养上清中TNF -α、IL - 6的释放情况 ,并检测人单核细胞株THP - 1细胞表面Toll样受体 9(TLR9)和Toll样受体 4 (TLR4 )表达以及核转录因子κB(NFκB)的活化情况。结果 :ECDNA可导致小鼠死亡 ,并具有明显的量效关系 ,小牛胸腺DNA和DNA酶消化的ECDNA则不能引起小鼠死亡 ;ECDNA与LPS均可导致大鼠血清TNF -α、IL - 6水平升高 ,变化趋势相似 ,但ECDNA所致的TNF -α峰值早于LPS组 1h出现。在体外实验中 ,不同浓度的ECDNA、LPS可诱导ANA - 1细胞大量释放TNF -α、IL - 6 ,以及THP - 1细胞表面TLR9和TLR4的高表达 ,但ECDNA诱导NFκB活化的能力远小于LPS。结论 :ECDNA可以和LPS一起参与SIRS的发生 ,可能机制与诱导单核细胞TNF -α、IL - 6的释放有关 ,但ECDNA诱导细胞因子释放的信号转导过程中可能尚有其它途径的存在。

AIM: To investigate whether the bacterial DNA participates in SIRS and its possible mechanism. METHODS: Escherichia coli genomic DNA (EC DNA) was extracted and purified from Escherichia coli 25922 by alkaline lysis method. Mortality of mice challenged with EC DNA and the changes of TNF-α and IL-6 in rat serum were observed. ANA-1 cells were cultured in vitro, after the cells were stimulated by different concentrations of EC DNA and LPS, the level of TNF-α and IL-6 in supernatant were tested. Meanwhile,expression of TLR9 and TLR4 on cell surface was measured. Activation of NF-κB was also observed. RESULTS: The lethal effect of EC DNA on mice with an obvious dose-effect relationship was observed. The death happened within 24 hours. Calf thymus DNA and DNase I-treated EC DNA did not lead to mice to die. The changes of serum TNF-α and IL-6 in rats induced by EC DNA and LPS were similar, but TNF-α peak level of EC DNA group appeared 1 hour earlier than that of LPS group. In vitro, large amount of TNF-α and IL-6 were released from ANA-1 cells stimulated by EC DNA. High expression of TLR9 and TLR4 was observed on surfaces of THP-1 cells. In particularly, LPS induced strong activation of NFκB. The results suggested other pathway possibly took part in the signal transduction inducea by EC DNA. CONCLUSION: EC DNA has the abilities to lead to death of mice, and induces serum TNF-α and IL-6 level to increase in rats and ANA-1 cells to release cytokines in vitro. High expression of TLR9 and TLR4, strong activation of NF-κB may be its important molecular mechanism, but other pathway probably exists to play an important role.