克雷伯杆菌分泌因子及菌体成分在细胞内、外刺激肺上皮细胞分泌IL-8的研究

Klebsiella pneumoniae secretory factors and sonicated bacterial extract inducing humanlung epithelial cells IL-8 secretion intracellularly and extracellularly

目的:通过比较肺炎克雷伯杆菌(Klebsiellapneumoniae,Kp)分泌因子及菌体成分对经过digitonin处理和未处理的肺上皮细胞株IL8分泌的影响,进一步了解克雷伯杆菌诱导肺上皮细胞炎症反应的信号转导机制。方法:实验分为两组,一组使用能使细胞膜通透性增加的温和去污剂digitonin处理,而另一组不处理。分别用Kp03183的细菌培养上清和超声处理的菌体成分刺激肺上皮细胞株A549,酶联免疫吸附实验(ELISA)检测细胞IL8表达水平。并用RT-PCR的方法检测肺上皮细胞胞内模式识别受体NOD1的表达。结果:Kp培养上清对未经digitonin处理的细胞IL8分泌无明显增强作用,与对照相比差异无显著意义(p>0.05),菌体成分能刺激IL8分泌增加(P<0.01),但增高不超过一倍。经digitonin处理使细胞膜通透性增加后,Kp培养上清及菌体成分刺激细胞IL8的作用都增强,菌体成分刺激IL8效果更为显著,为对照的3倍,而培养上清作用相对较弱。RT-PCR检测结果表明,肺上皮细胞表达胞内模式识别受体NOD1。结论:菌体成分是诱导炎症反应更有效的刺激物,肺炎克雷柏杆菌侵入肺上皮细胞可能是引发细胞炎症反应的始动环节。肺上皮细胞表达胞内模式识别受体NOD1,它是否参与肺上皮细胞识别肺炎克雷伯杆菌菌体成分,值得进一步的研究。

Objective: To investigate the mechanisms of inflammation signal transduction induced by Klebsiella pneumoniae in human lung pithelial cells by comparing the IL-8 expression level between digitonin-treated and untreated A549 cells stimulated by secretory factors and the sonicated bacterial extract of Klebsiella pneumoniae. Methods: Human lung epithelial cell line A549 treated with or without digitonin were stimulated by bacterial supernatant and sonicated bacterial extract from Klebsiella pneumoniae respectively. The expression level of IL-8 was detected using ELISA. The intracellular pattern recognition receptor NOD1 is detected in human lung epithelial cells by RT-PCR. Results: Bacterial supernatant showed no significantly effect on the IL-8 expression level in A549 cells without the addition of digitonin, whereas there was a slightly increase in the expression of IL-8 in digitonin-treated cells. Sonicated bacterial extract caused a modest, less than 1 fold, increase in IL-8 secretion by digitonin-untreated A549 cells. In the digitonn-permeabilized A549 cells, Sonicated bacterial extract strongly induced the expression of IL-8, a 3 fold increase over the control. Finally, the expression of nod1 mRNA in human lung epithelial cells is proved by RT-PCR. Conclusion: Sonicated bacterial extract from Klebsiella pneumoniae is a more effective inflammation stimulator to lung epithelial cells than bacteria secretory factors. The entry of the bacteria into the cells may be necessary for Klebsiella pneumoniae to induce effectively inflammatory response in vivo in the lung epithelial cells.