Functional Analysis of Antimicrobial Peptides in Protection from Bacterial Infection

最初，我们研究了可诱导HBD在体外肺炎支原体感染急性期的作用。我们观察到，化学合成的HBD-2和HBD-3，而不是HBD-1，对肺炎支原体显示出有效的抗菌活性。为了诱导HBD的产生，将人肺鳞状细胞系EBC-1与强大的刺激剂IL-1β孵育。检测EBC-1细胞培养上清和总RNA中HBD的表达。Western印迹分析检测培养上清液中HBD-2的表达。IL-1bβ可显著上调Hbd-2m RNA的表达，但对Hbd-1和Hbd-3的表达无明显影响。用IL-1β处理的EBC1细胞感染肺炎支原体，以确定HBD是否具有抗感染作用。集落实验显示，IL-1、β处理的EBc-1对肺炎支原体的生长有明显的抑制作用。因此，我们的结果表明，IL-1β处理的EBc-1细胞产生的HBD-2对肺炎支原体感染的早期阶段具有保护作用。随后，在体内感染模型中检测了抗菌肽，特别是JAMP的作用。似乎没有关于抽筋和支原体感染的报道。用化学方法合成了一种化学合成的杀菌素类化合物。该抽筋对肺炎支原体有较强的抗菌活性。用肺炎支原体鼻腔感染BALB/c小鼠，分别于感染后6、12、24、48h取感染肺组织，观察其在体内的痉挛表达情况。免疫印迹分析表明，感染后24小时从肺组织中提取的匀浆中含有痉挛。另外，用RT-PCR方法检测了感染肺组织中JRAMP基因的表达。免疫组织化学染色显示，支气管区有大量抽筋阳性的中性粒细胞，但未见明确的抽筋阳性的上皮细胞。目前正在调查抽筋阳性中性粒细胞与保护作用之间的关系。

Initially, we examined the role of inducible hBD in the acute phase of in vitro M.pneumoniae infection. We observed that chemically synthesized hBD-2 and hBD-3, but not hBD-1, show effective antimicrobial activity against M.pneumoniae. To induce hBD production, a human pulmonary squamous cell line EBC-1 was incubated with a potent stimulant, IL-1β. Culture supernatant and total RNA from the EBC-1 were examined for hBD expression. hBD-2 in the supernatant was detected by western blot analysis. In addition, hBD-2 mRNA was strongly up-regulated by IL-1bβ, but neither hBD-1 nor hBD-3 was apparently up-regulated. To determine whether hBD induces the protection from infection, IL-1β-treated EBC-1 cell were infected with M.pneumoniae. IL-1β-treated EBC-1 significantly inhibited the growth of M.pneumoniae, as judged by colony assay. Thus, our results suggest that hBD-2 produced by IL-1β-treated EBC-1 cells plays a role in the protection of early stage of M.pneumoniae infection.Subsequently, roles of antimicrobial peptides, in particular CRAMP, in in vivo infection model were examined. There seems to be no reports regarding the CRAMP and Mycoplasma infection. CRAMP, a family of cathelicidin, was chemically synthesized. The CRAMP showed an effective antimicrobial activity against M.pneumoniae. To induce CRAMP in vivo, BALB/c mice were intranasally infected with M.pneumoniae, and infected lungs taken from the mice at 6,12,24 and 48 hours postinfection were examined for CRAMP expression. Western blot analysis showed that the homogenate from the lungs at 24 hours after infection contains CRAMP. In addition, CRAMP mRNA from the infected lungs was detected by using RT-PCR. Immunostaining showed that there are many CRAMP-positive neutrophils in the peribronchial area, but no definitive CRAMP-positive epithelial cells. At present underway is investigation on the association between CRAMP-positive neutrophils and protective effect