重组防御素5对铜绿假单胞菌的杀菌效应和抗粘附作用

Bactericidal and anti-adherent effects of recombinant human defensin 5 on Pseudomonas aeruginosa

目的:观察重组防御素5(rHD-5)对铜绿假单胞菌的杀菌/抑菌效果。方法:将冷存的 rHD-5按照浓度稀释成100μg/ml(Ⅰ液)、200μg/ml(Ⅱ液)和300μg/ml(Ⅲ液),以不含 rHD-5的磷酸盐缓冲液(O 液)为对照,观察了它们对大肠杆菌标准株(ATCC25922)、铜绿假单胞菌标准株(ATCC27853)、铜绿假单胞菌野生株的杀菌效果,以及抗铜绿假单胞菌野生株对小肠上皮细胞(IEC-6)粘附的效果。结果:随着 rHD-5浓度的逐渐增加,对大肠杆菌的抑菌坏直径也相应扩大,O 液、Ⅱ液、Ⅲ液的抑菌坏直径分别为0mm、15.78±0.67mm、31.11±0.89、36.94±0.77mm;虽然对铜绿假单胞菌标准株的抑菌坏直径没有上述趋势,但Ⅲ液产生了抑菌环,直径为16.67±0.79mm,对铜绿假单胞菌野生株则无抑茵环产生。此外,O 液、Ⅰ液、Ⅱ液、Ⅲ液干预铜绿假单胞菌野生株后,对 IEC-6的平均粘附率分别为98.02%±0.51%、18.68%±1.49%、9.46%±2.74%、0.73%±0.44%,单个 IEC-6细胞所粘附的细菌数分别为20.52±6.70、4.26±2.70、6.88±3.92和0.68±0.79,受损害的 IEC-6所占百分比分别为30.88%±30.48%、8.36%±4.52%、2.94%±3.58%、0%,均呈剂量依赖性降低趋势。结论:rHD-5对铜绿假单胞菌的杀菌及抑菌作用,提示其在防治肠源性感染中具有潜在的临床应用价值。

Objective:To observe bactericidal and anti-adherent effects of recombinant human defensin 5 (rHD-5) on Pseudomonas aeruginosa.Methods:100,200,300μg/ml concentrations of recombinant human defensin 5 (HD5) (Solution Ⅰ,Ⅱ,Ⅲ) were diluted and obtained by lyophilization from CHO-K1 ceil culture supernatant,which were transferred by recombinant plasmids (pcDNA3.1(+)-DEFA5).The untransferred CHO-K1 cell culture su- pernatant (solution O) served as control.Bactericidal effects of the above-mentioned four solutions were observed by bactericidal test involving Escherichia coli (ATCC 25922),P.aeruginosa (ATCC 27853),and wild P.aerugi- nosa.The anti-adherent activity of the solutions to wild P.aeruginosa on intestinal epithelial ceil line (IEC-6) was also studied.Results:Diameters of inhibition zone of the solutions (O,Ⅰ,Ⅱ,Ⅲ) to E.coli (ATCC 25922) in- creased along with the increase in rHD-5 concentration,measuring 0 mm,15.78±0.67mm,31.11±0.89mm and 36.94±0.77 mm respectively.Although no such bactericidal tendency against P.aeruginosa (ATCC 27853) and wild P.aeruginosa,inhibition zone in P.aeruginosa (ATCC 27853) culture was observed with solution Ⅲ(16.67 ±0.79mm).No bactericidal effect of them was found in wild P.aeruginosa.The average adherent rates of them (Solution O,Ⅰ,Ⅱ,Ⅲ) to wild P.aeruginosa were 98.02%±0.51%,18.68%±1.49%,9.46%±2.74%,and 0.73%±0.44%,respectively.The number of P.aeruginosa adhered to IEC-6 were 20.52±6.70,4.26±2.70, 6.88±3.92,and 0.68±0.79,respectively.The rates of damaged IEC-6 cells were 30.88±30.48%,8.36%± 4.52%,2.94%±3.58%,and 0,respectively.Obviously,all of the three indexes were decreased parallel to the increase in rHD-5 concentration.Conclusions:The results of bactericidal and anti-adherent activity of rHD-5 to P. aeruginosa indicate that there is a potential value of rHD-5 in the prevention and treatment of endogenous infec- tion.