内毒素结合肽P1和P2抗内毒素活性的实验研究

摘要

Objective To study the antiendotoxin activity of P1 and P2 based on the lipopolysacchride binding protein.Method P1 and P2 were designed and obtained.In vitro test,peripheral blood mononuclear cell(PBMC)were extracted from volun-teer 100ml venous blood,the experiment group was arranged as:control group,positive control group,LPS group,LPS+P1 (2 mg/L,5 mg/L,12.5 mg/L)group and LPS+P2(2 mg/L,5 mg/L,12.5 mg/L)group,TNF-α and IL-6 of supernatant liquor in every group were detectd by ELISA.In vivo,40 kunming rice were randomly divided into four groups with ten rice each group:control group,model group,LPS+P1 and LPS+P2 group,Pathologic changes of lung and liver tissues were ob-served by hematoxylin and eosin(HE)staining.Results The serum level of IL-6 and TNF-αin 12.5 mg/L P1 and 2 mg/L, 5 mg/L,12.5 mg/L P2 treatment group were lower than that in model group,the difference was statistically significant(all P<0.01).Serum level of TNF-αor IL-6 in 12.5 mg/L P2 treatment group were similar to that in PMB treatment group, and there was no statistically significant difference(P>0.05).Histologymorphology finding showed that central veins of liver and hepatic sinusoid congestion,hepatic cellular edema existed,occasionally,acidophilic change and spotty necrosis were found,pulmonary interstitial edema,focal hemorrhage,alveolar space stenosis existed.As regards 10 mg/kg P1 treatment group mice,hepatic cellular edema and pulmonary interstitial edema ameliorated.About 10 mg/kg P2 treatment group mice, veins of liver and hepatic sinusoid congestion obviously ameliorated,mild pulmonary interstitial edema exsited.Conclusion The results indicated P1 and P2 had antiendotoxin effect,in vivo and vitro,for 12.5 mg/L P2,its inhibition effect for TNF-αor IL-6 release was positive.%目的 研究基于脂多糖(lipolysachride,LPS)结合蛋白的新型内毒素结合肽 P1和 P2的抗内毒素活性.方法 设计并合成内毒素结合肽P1和P2.体外实验取一名健康志愿者静脉血100 ml分离的外周血单个核细胞(PBM),分为正常对照组、LPS组、阳性对照组 LPS(1 mg/L)+多黏菌素 B(12.5 mg/L)、P1组 LPS(1 mg/L)+P1(2 mg/L,5 mg/L,12.5 mg/L)、P2组 LPS(1 mg/L)+P2(2 mg/L,5 mg/L,12.5 mg/L).ELISA方法检测各组上清液中 TNF-α和 IL-6的浓度.体内实验将40只昆明小鼠平均分为四组,每组10只,分别为空白对照组、LPS模型组、LPS+P1治疗组和 LPS+P2治疗组.取各组小鼠肺脏和肝脏组织制作 HE切片,观察组织形态学变化.结果 ①与1 mg/L LPS比较,1 mg/L LPS+12.5 mg/L的P1以及1 mg/L LPS+3种浓度P2刺激后,PBMC培养上清液中 IL-6及TNF-α的水平均明显降低,差异均有统计学意义(均P<0.01),12.5 mg/L的P2与阳性对照 PMBC作用后上清液中 TNF-α浓度比较,差异无统计学意义(P>0.05).②组织形态学显示,模型组小鼠肝小叶中央静脉及肝窦扩张充血,肝细胞浊肿,偶见肝细胞嗜酸性变及点状坏死.肺间质充血水肿,灶性出血,肺泡腔狭小.10 mg/kg P1处理组肝细胞浊肿较模型组减轻,肺间质充血水肿减轻.10 mg/kg P2处理组,肝小叶中央静脉及肝窦充血程度减轻,肺间质充血水肿明显减轻.结论 体内外实验表明合成内毒素结合肽P1和P2对 LPS导致的机体损伤具有拮抗作用.体外实验结果还表明,12.5 mg/L P2对TNF-α或 IL-6的释放抑制作用较为肯定.