摘要
目的探讨Elafin重组气道上皮细胞对铜绿假单胞菌(Pseudomonas aeruginosa,Pa)生物被膜(biofilm,BF)的抗菌作用。方法体外平板法制备Pa生物被膜模型,用银染法及扫描电镜鉴定。体外培养A549细胞,将pEGEP-N1-Elafin重组到A549细胞,倒置荧光显微镜观察转染情况。采用猪胰弹性蛋白酶(NE组)、铜绿假单胞菌(Pa组)上清、大肠埃希杆菌(E．coli组)上清分别诱导孵育重组细胞24 h,另设转染未含Elafin基因空载体的A549细胞作为对照组。将成熟BF载体放入4组中继续孵育8 h,扫描电镜观察4组BF结构的变化,并于第8小时用四甲基偶氮唑盐(MTT)法测定细菌存活数。结果扫描电镜下观察到3个诱导组的BF结构较对照组的BF明显松散,尤以Pa组和NE组为甚;NE组[(3．238±0．316)×106 CFU／cm2]、Pa组细菌数量[(3．317±0．247)×106 CFU／cm2]均较对照组[(5．946±0,453)×106 CFU／cm2]显著减少(P<0．01),E．coli组细菌数量[(5．138±0．391)×106 CFU／cm2]较Pa组和NE组减少,差异均有统计学意义(P<0．01)。3个诱导组BF细菌清除率与对照组相比分别为45．6％、44．2％和13．6％。结论Elafin重组气道上皮细胞能有效清除Pa生物被膜中的细菌,而Pa产物也是Elafin表达的有效诱导物。
Objective To explore whether airway epithelial cells（A549 cells）after transfected by elafin have antimicrobial effect on P. aeruginosa （Pa） biofilm. Methods To estabhsh the Pa biofilm model in vitro, and proved by a rapid silver nitrate staining procedure and scanning electronic microscope （SEM）. After cultivating the cells, the constructed pEGFP-N1-Elafin eukaryotic expression vector has transfected into A549 cells by LipofectAMINE^TM2000. Elafin transfected cells incubated by pig pancreas elastase （NE group）, the supematant of Pa （ Pa group） and Eseherichia eoli （ E. eoli group） respectively for 24 h. A549 cells transfeeted the vector without elafin gene as control group. After biofilm carriers put into each group and incubated for 8 h, we observed the structure of biofilm in each group by SEM, and measured the number of each group by MTF at 8 h. Results Both silver nitrate staining procedure and SEM demonstrated bacterial biofilm. The structure of BF in three groups was changed. The number of bacteria in both NE group [ （3. 238 ± 0. 316） ×10^6CFU/cm^2] and Pa group [ （3. 317 ± 0.247）×10^6CFU/cm^2] was decreased as compared to the control group [（5.946±0.453）×10^6CFU/cm^2]. The ratio of bacteria cleating in Pa group（44.2%） and NE group （45.6%）was higher than that of in E. coli group（13.6%）. Conclusion The airway epitheha transfected by elafin resist and clean Pa bioflim, meanwhile, the products of Pa are effective inducing factors on the expression of elafin.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第8期725-728,共4页
Chinese Journal of Microbiology and Immunology
