铜绿假单胞菌群体感应系统在生物膜感染中对调节性T细胞的影响

发布时间：2018-02-05 19:39

本文关键词： 铜绿假单胞菌生物膜群体感应系统调节性T细胞　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨铜绿假单胞菌的群体感应系统在生物膜感染中对宿主的调节性T细胞的影响。方法:选取铜绿假单胞菌的三个菌株即野生株PAO1和两个QS系统的基因缺陷株△lasI△rhlI,△lasR△rhlR采用体外静态模型培养3天获得成熟生物膜,收集生物膜上清液(BCM)。三个细菌菌株的BCM分别与人外周血单个核细胞(PBMCs)共通过培养16 h,用CCK-8法检测BCM的细胞毒性。细胞悬液离心,其中的细胞提取总RNA,定量PCR检测Foxp3的mRNA水平,而上清液用ELISA法检测TGF-β1,IL-10以及IL-6的蛋白水平。另外,采用流式细胞术检测调节性T细胞。建立大鼠生物膜感染模型,检测Tregs相关因子Foxp3和TGF-β1,动物实验验证QS系统和调节性T细胞相关性。结果:PBMCs经BCM处理后,PAO1-BCM,△lasR△rhlR-BCM及△lasI△rhlI-BCM三个处理组均表现出明显的细胞毒性。PAO1-BCM处理组Foxp3 mRNA表达量(1.15±0.16)较△lasI△rhlI-BCM及△lasR△rhlR-BCM处理组(0.97±0.110.93±0.12)明显升高(P0.05),但是两个QS系统缺陷菌株BCM处理组之间无统计学差异。而且PAO1-BCM组TGF-β1的蛋白表达水平(844.49±40.97 pg/ml)较△lasI△rhlI-BCM及△lasR△rhlR-BCM组的表达水平(725.69±129.16pg/ml740.29±35.86 pg/ml)明显升高(P0.05),类似的两QS系统缺陷菌株的BCM处理组之间比较无统计学差异。但是值得注意的是,PAO1-BCM组IL-10的表达水平(2.46±1.22 pg/ml)较△lasI△rhlI-BCM及△lasR△rhlR-BCM组的表达水平(33.29±6.11 pg/ml35.11±7.72pg/ml)却明显减低(P0.05)。而△lasI△rhlI-BCM及△lasR△rhlR-BCM两个处理组(799.27±75.66 pg/ml,1305.42±198.48 pg/ml)的IL-6的蛋白表达量较PAO1-BCM处理组(12.63±8.31 pg/ml)明显升高。动物实验中发现Foxp3,TGF-β1的变化与体外实验的结果一致。结论:铜绿假单胞菌QS系统lasI/rhlI基因和lasR/rhlR基因在铜绿假单胞菌生物膜感染中可促进宿主的调节性T细胞的活化,这可能是最终导致临床上生物膜感染持续性和慢性化的一个机制。
[Abstract]:Objective: to investigate the effect of colony sensing system of Pseudomonas aeruginosa on host regulatory T cells in biofilm infection. Three strains of Pseudomonas aeruginosa, that is, wild strain PAO1 and two QS gene-deficient strains, lasI rhlI were selected. The mature biofilm was obtained by lasR rhlR culture in vitro static model for 3 days. The BCM of three bacterial strains were co-cultured with human peripheral blood mononuclear cells (PBMC) for 16 h. The cytotoxicity of BCM was detected by CCK-8 method. The cell suspension was centrifuged. The total RNAs were extracted from the cells and the mRNA level of Foxp3 was detected by quantitative PCR. The protein levels of TGF- 尾 1 IL-10 and IL-6 in supernatant were detected by ELISA method. In addition, the regulatory T cells were detected by flow cytometry, and the model of biofilm infection in rats was established. Tregs related factors Foxp3 and TGF- 尾 1 were detected, and the correlation between QS system and regulatory T cells was verified by animal experiments. LasR rhlR-BCM and lasI rhlI-BCM showed obvious cytotoxicity. PAO1-BCM treatment group Foxp3. MRNA expression (. 1.15 卤0.16) was significantly higher than that of lasI rhlI-BCM and lasR rhlR-BCM (0.97 卤0.110.93 卤0.12) (P < 0.05). P0.05). However, there was no significant difference between the two QS system-deficient strains treated with BCM, and the protein expression level of TGF- 尾 1 in PAO1-BCM group (. The expression level of 844.49 卤40.97 PG / ml was higher than that of lasI rhlI-BCM and lasR rhlR-BCM (P < 0.05). 725.69 卤129.16 PG / ml 740.29 卤35.86 PG / ml increased significantly (P 0.05). There was no statistical difference between the BCM treatment groups of similar QS system deficient strains, but it was worth noting. Expression of IL-10 in PAO1-BCM group: 2.46 卤1.22 PG / ml). The expression level of lasI rhlI-BCM and lasR rhlR-BCM was 33.29 卤6.11 pg/ml35.11 卤7.72 PG / ml. However, the P0. 05 pg/ml was significantly decreased in lasI rhlI-BCM and lasR rhlR-BCM treatment groups (799.27 卤75.66 pg/ml). The protein expression of IL-6 in 1305.42 卤198.48 PG / ml group was higher than that in PAO1-BCM treatment group (12.63 卤8.31 PG / ml). Foxp3 was found in animal experiments. The changes of TGF- 尾 1 were consistent with the results of in vitro experiments. The QS system lasI/rhlI gene and lasR/rhlR gene of Pseudomonas aeruginosa can promote the activation of host regulatory T cells in biofilm infection of Pseudomonas aeruginosa. This may be a mechanism that ultimately leads to the persistence and chronicity of biofilm infection in clinic.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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