粪肠球菌生物膜形成过程中群体感应相关基因的表达研究  

Expression of quorum-sensing related genes during Enterococcus faecafis biofilm formation

作　　者：卢志山[1] 孟磊[1] 柳忠豪[1] 任光辉[1] 孙爱杰[1] 刘湘梅[1] 

机构地区：[1]滨州医学院附属烟台市口腔医院牙体牙髓科,山东烟台264008

出　　处：《中华口腔医学杂志》2013年第8期485-489,共5页Chinese Journal of Stomatology

摘　　要：目的研究粪肠球菌生物膜形成过程中群体感应相关基因的表达情况，探讨其与生物膜形成的相关性。方法体外建立粪肠球菌生物膜模型，激光共聚焦显微镜观察6、12、24、48h生物膜的形成情况，结晶紫染色观察粪肠球菌生物膜形成的量，提取各时期生物膜细菌样本，通过实时荧光定量PCR对粪肠球菌生物膜形成过程中群体感应相关基因fsrB、明胶酶和丝氨酸蛋白酶（serine protease，sprE）的表达进行检测。结果粪肠球菌生物膜细菌分布不均匀，由死细菌和活细菌组成，6h时粪肠球菌生物膜细菌初始黏附，24h时生物膜基本成熟；24h内粪肠球菌生物膜的量随时间增加而增大，在培养0、6、12、24h时生物膜的量（4值）分别为0．00±0．00、1．09±0．13、2．10±0．79及3．30±0．13，两两比较差异均有统计学意义（P〈0．05）；48h生物膜的量（3．51±0．01）与24h生物膜的量相比略有增加，但差异无统计学意义。粪肠球菌生物膜形成过程中群体感应相关基因fsrB在0、6、12、24h时的表达量（2-ΔCT值）逐渐增大，分别为9．98±0．46、23．45±1．13、47．30±2．49及331．30±2．18，两两比较差异均有统计学意义（P〈0．05）；48h粪肠球菌生物膜内fsrB的表达量（11．0±0．39）与0h相比差异无统计学意义。明胶酶在0、6、12、24h时的表达量（2±值）逐渐增大，分别为6．54±0．73、14．26±1．24、37．47±2．35及264．80±5．10，两两比较差异均有统计学意义（P〈0．05）；48h粪肠球菌生物膜内明胶酶的表达量（9．09±0．81）与0h相比差异无统计学意义。sprE在0、6、12、24h时的表达量（2-ΔCT值）逐渐增大，分别为7．72±0．74、21．15±0．96、49．87±3．18及441．89±7．74，两两比较差异有统计学意义（P〈0．05）；48h粪肠球菌生物膜内sprE的表达量（11．04±1．74）与0h相比差异无统计学意义。结论粪�Objective To investigate the relationship between the expression of the quorum-sensing related genes during Enterococcus faecalis （ Ef ） biofilm formation. Methods Ef biofilms model was established in vitro and film formation process was observed by confocal laser scanning microscope at 6, 12, 24 and 48 hours respectively. Quantification of biofilms was achieved by staining with crystal violet. Real-time fluorescence quantitative PCR method was used to detect the expression of fsrB, gelE and sprE genes in the process of Ef biofilm formation. Results A lot of live and dead bacteria unevenly distributed in Ef biofilm. The quantity of biofilms increased with time within 24 hours and was 0 h ：0. 00 ± 0. 00,6 h： 1.09 -± 0. 13,12 h：2. 10 ±0. 79,24 h：3.30 ±0. 13, which was significantly different among the 4 time period（P 〈 0. 05）. The quantity of biofilm at 48 h（3.51 ±0. 01 ） increased slightly compared with 24 h（3.30 -±0. 13）, but did not show significant difference. Quantitative real-time PCR showed that the expression of quorumsensing related fsrB increased with time within 24 hours and was 0 h：9.98 ±0.46,6 h：23.45 ± 1.13,12 h： 47.30 ±2.49,24 h： 331.30 ±2. 18, which was significantly different among the 4 time period（P 〈0. 05）. The expression of gelE was 0 h： 6. 54 ±0. 73,6 h： 14. 26 ± 1.24,12 h： 37.47 ±2. 35,24 h：264. 80 ±5.10 （ P 〈 0. 05 ）. The expression of sprE was 0 h ： 7.72 ± 0. 74,6 h ： 21.15 ± 0. 96,12 h ： 49.87 ± 3.18,24 h ： 441.89 ± 7.74, which was significantly different among the 4 time period （ P 〈 0. 05 ）. Conclusions The fsrB, gelE and sprE genes are closely related to the biofilm formation in Ef.

关 键 词：肠球菌 粪 生物膜 群体感应 基因表达 

分 类 号：R378[医药卫生—病原生物学]