鲎抗内毒素因子模拟肽4的体外生物活性实验研究

Endotoxin-neutralizing activity of modeling peptide 4 from the Limulus anti-lipopolysaccharide factor (M4) in vitro

目的研究鲎抗内毒素因子模拟肽4(m ode ling peptide 4 from the L im u lus an ti-lipopo lysaccharide factor,M 4)体外中和内毒素的活性。方法研究以对内毒素高亲和力的多粘菌素B(po lym yx in B,PM B)为对照,M 4设80、40、20、10、5μm o l/L浓度,结合内毒素的活性以生物传感技术分析;中和内毒素的能力以鲎试验动态浊度法测定;对LPS刺激THP-1细胞释放TNF-α的影响采用EL ISA试剂盒定量观察。结果M 4具有较好的内毒素亲和力和中和活性,在5、10、20、40和80μm o l/L浓度时对内毒素2EU/m l(1EU约等于100pg)的中和率分别为43.44%、47.76%、63.12%、81.54%和84.54%,呈明显的浓度梯度效应,20μm o l/L以上浓度时与PM B作用相当。对THP-1细胞释放TNF-α的影响结果显示,M 4可明显抑制LPS刺激的TNF-α释放,但M 4本身具有诱导THP-1细胞释放TNF-α的作用。结论M 4具有较强的中和内毒素活性,值得进一步深入研究。

Objective To study the endotoxin-neturalizing activity of modeling peptide 4 from the Limulus anti-lipopolysaccharide factor (M4) in vitro. Methods The endotoxin-affinity of M4 was analyzed by biosensor technique while the endotoxin-neutralizing capability was determined by Limulus amebocyte lysate test. The high affinity endotoxin-binding peptide polymyxin B (PMB) was used as control. The effect of M4 on the human monocytic leukemia (THP-1) cell producing TNF-α induced by LPS (lipopolysaccharide) was (examined) by ELISA Kit. Results M4 showed a good endotoxin-binding and neutralizing activity. To the 2EU/ml LPS, the neutralizing rate of M4 at 5μmol/L, 10μmol/L, 20μmol/L, 40μmol/L and 80μmol/L were observed to be 43.44%, 47.76%, 63.12%, 81.54% and 84.54%, respectively, which presented a clear dose-effect relationship. The endotoxin-neutralizing effects of M4 at concentrations over 20μmol/L were seemed to be the same as that of PMB. Furthermore, the results of TNF-α release of THP-1 cell showed that M4 itself had an ability to induce TNF-α production but could inhibit the TNF-α release stimulated by LPS remarkably. Conclusion M4 had a potent endotoxin-binding and neutralizing effects and deserved further study.