内生多肽Elafin转染气道上皮细胞对不同细菌生物膜作用的差异

Different effects of airway epithelial cells transfected by elafin on bacterial biofilm

目的探讨内生多肽(Elafin)转染气道上皮细胞经不同细菌产物诱导后对铜绿假单胞菌(P.aeruginosa,Pa)生物膜(biofilm,BF)的效应差异。方法将pEGFP-N1-elafin转染到培养的肺腺癌细胞株A549细胞后,用表皮葡萄球菌(表葡组)、大肠埃希杆菌(E.coli组)和铜绿假单胞菌(Pa组)培养上清分别孵育细胞24h,另设转染空载体A549细胞作为正常组。用ELISA和Western blot检测每组细胞Elafin分泌水平和细胞内含量;体外平板法制备Pa-BF模型,用快速银染法和扫描电镜鉴定;将BF载体放入上述4组细胞中继续孵育8h,银染法观察BF中每个视野细菌的(灰度)面积积分,扫描电镜观察形态学改变。结果Pa组细胞Elafin分泌水平和细胞内含量均较正常组的明显升高(P<0.01),E.coli组也有升高(P<0.05),表葡组无明显变化;各组BF面积积分也显示有明显差异,Pa组、E.coli组BF结构较正常组明显松散,尤以Pa组为甚,表葡组则没有明显变化。结论不同细菌对Elafin的诱导表达能力有差异:Pa较强,E.coli次之,表葡不明显;Elafin能清除生物膜细菌。

Objective To explore the different effect of elafin incubated by different bacteria on P. aeruginosa （Pa） bioflim. Methods To cultivate the A549 cells in vitro, the pEGFP-N1-elafin eukaryotic expression vectors have been transfected to the cells by LipofectamineTM 2000. Elafin transfected cells incubated by the supernatant of S. epidermidis （ S. epidermidis group）, Pa （ Pa group） and E. coli （E. coli group） respectively for 24 hours, the A549 cells were transfected. Then the levels of elafin were detected by ELISA and Western blot. To establish the Pa biofilm model in vitro, a rapid silver nitrate staining procedure and scanning electronic microscope （SEM）demonstrated bacterial biofilm. After biofilm carriers were put into each group and incubated for 8 hours, we measured the proportion of bacteria biofilm by silver nitrate staining and observed the structure of biofilm by SEM. Results The Pa and E. coli groups （especially Pa） raised the content of elafin in cells and the level of secretion increasing as compared to the normal group, while the S. epidermidis group had no change. Both silver nitrate staining procedure and SEM demonstrated the prestnce of bacterial biofilms. The the proportion of bacteria biofilm and the structure of BF in Pa and E. coli groups were changed, especially in Pa group. Condusion There was a specificity for bacteria to induce the express of elafin. The inducing effect of Pa was more significant than that of E. coll.