基于多肽阵列技术探讨芪归银方对多重耐药铜绿假单胞菌感染大鼠的干预作用

Based on Polypeptide Array Technique to Explore the Intervention of Qiguiyin Decoction on Rats Infected by Multidrug Resistant Pseudomonas Aeruginosa

目的利用多肽阵列技术探讨芪归银方对多重耐药铜绿假单胞菌感染大鼠抗原抗体反应的影响。方法雄性SD大鼠36只,采用随机数字表法分为空白组、模型组、芪归银方组共3组各12只。参考文献建立多重耐药铜绿假单胞菌腹腔感染大鼠模型。采用多肽阵列检测各组大鼠血清对金属酶VIM-1、SPM-1和超广谱内酰胺酶TEM-13种具有代表性的耐药蛋白抗体表达,比较各组之间的差异。结果空白组大鼠血清对耐药蛋白VIM-1的重叠多肽片段产生不同程度的抗体反应,而铜绿假单胞菌感染后,血清抗体反应的明显变化表明,多肽片段指征的耐药蛋白VIM-1的抗原决定簇能够激发大鼠的免疫反应。芪归银方组大鼠中,针对表位7~11和36-40的抗体表达强度大于空白和模型组的抗体表达强度(均P<0.05);模型组中,针对53-54和56-58表位的抗体反应强度降低(均P<0.05),而芪归银方组的抗体反应虽较正常组为低,但高于模型组大鼠(P<0.05);与空白组比较,模型组和芪归银方组大鼠均产生针对23-25表位的较强抗体反应,两者之间比较差别不大(P>0.05);而模型组对80-81表位产生显著的抗体反应,但在空白组和芪归银方组中则无明显反应(均P>0.05)。空白组大鼠血清对耐药蛋白SPM-1的重叠多肽片段产生不同程度的抗体反应,但各组间差别不大(均P>0.05)。感染刺激大鼠产生新的更多地针对13-15和39-40表位的抗体,然而,相较于空白组和芪归银方组产生的针对16-19和82-85抗原的免疫反应,模型组大鼠则产生的抗体反应更弱(P<0.05)。铜绿假单胞菌感染后刺激大鼠免疫系统并且产生更强的针对多肽19-21、26-27、52-55和66-70的反应。3组大鼠血清中抗体针对的阳性表位数目差别不大(均P>0.05);芪归银方组对TEM-1蛋白19-21和52-55表位的抗体反应强于模型组和空白组(均P<0.05)。结论芪归银方能够通过刺激感染后大鼠产生抗耐药蛋白VIM-1、SPM-1和TEM-1的保护性抗体,达到提高多重耐药铜绿假单胞菌感染大鼠免疫应答的目的。

Objective:To investigate the effects of Qiguiyin Decoction on antigen and antibody reaction of rats infected with multi-drug resistant Pseudomonas aeruginosa by polypeptide array technique. Methods:36 male SD rats were randomly divided into the blank group,the model group and Qiguiyin Decoction group,12 rats in each. Reference was taken to establish rat models of intraperitoneal infection by multidrug-resistant Pseudomonas aeruginosa. Polypeptide arrays were used to detect the expression of three representative drug-resistant proteins antibody,vim-1,SPM-1 and TEM-1,and to compare the differences among the groups. Results:The serum of the blank group showed different levels of antibody response to the overlapping polypeptide fragments of the drug-resistant protein VIM-1. The changes of serum antibody response after Pseudomonas aeruginosa infection showed that the antigenic determinants of the drug-resistant protein VIM-1 indicated by the polypeptide fragments on the upper surface could stimulate the immune response of the rats. In Qiguiyin Decoction group,the antibody expression intensity of epitopes 7~11 and 36~40 was higher than that of the blank group and the model group(all P < 0.05);in the model group,the antibody reaction intensity of epitopes 53-54 and 56-58 was lower(all P < 0.05),while in Qiguiyin Decoction group,the antibody reaction was lower than that of the normal group,but higher than that of the model group(P < 0.05). Compared with the blank group,the model group and Qiguiyin Decoction group all produced strong antibody response to epitope 23-25,and there was no significant difference between them(P > 0.05);while the model produced significant antibody response to epitope 80~81,but there was no significant reaction in the blank group and Qiguiyin Decoction group(P > 0.05). The serum of the blank group produced antibody responses to the overlapping polypeptide fragments of SPM-1,but there was no significant difference among the three groups(all P > 0.05). Infection stimulated rats to produce new and more antibodies targeting epitopes 13~15 and 39~40. However,compared with the blank group and Qiguiyin Decoction group,the immune response to antigens 16~19 and 82~85 was weaker in the model group(P < 0.05). Pseudomonas aeruginosa stimulated the immune system of rats and produced stronger responses to polypeptides 19-21,26-27,52-55 and 66-70. The number of positive epitopes of TEM-1 protein 19-21 and 52-55 in Qiguiyin Decoction group was higher than that in the model group and the blank group (all P < 0.05). Conclusions:Qiguiyin Decoction can improve the immune response of rats infected with multi-drug-resistant Pseudomonas aeruginosa by stimulating the production of protective antibodies of VIM-1,SPM-1 and TEM-1.