Ling Zhou, Dayong Chen, Denggao Yu, Yiting Wang, Liaoqiong Fang, Zhibiao Wang
State Key Laboratory of Ultrasound Engineering in Medicine Co-Founded by Chongqing and the Ministry of Science and Technology, College of Biomedical Engineering, Chongqing Medical University, Chongqing, China (mainland).
Med Sci Monit Basic Res. 2019 Feb 11;25:45-52. doi: 10.12659/MSMBR.913756.
BACKGROUND Recent studies have shown that Escherichia coli induced digestive tract diseases may be related to outer membrane vesicles (OMVs) induced intestinal double-strand breaks (DSBs) in intestinal epithelial cells. This study aimed to compare the impact of OMVs forces on DSBs in intestinal epithelial Caco-2 cells, and provide a new treatment for digestive diseases caused by E. coli. MATERIAL AND METHODS E.coli OMVs were prepared and co-cultured with Caco-2 cells. The uptake of OMVs by Caco-2 cells was observed by confocal microscopy. The γ-H2AX protein was detected by western-blots. The DSBs caused by OMVs was detected by single cell gel electrophoresis. RESULTS The particle size analyzer showed that the average diameters of OMVs centrifuged at 20 000×g and 50 000×g were 217.5±7.29 nm and 186.3±6.59 nm (P<0.05), respectively. Transmission electron microscopy of the OMVs revealed a lipid bilayer structure with a variety of different sizes. Confocal fluorescence microscopy revealed that OMVs almost completely entered Caco-2 cells after 24 hours. The ratio of γ-H2AX protein band gray value normalized data in the OMVs centrifuged at 20 000×g and 50 000×g, and the control group (without OMVs) were 2.23±0.18, 1.58±0.20, 1±0.30 (P<0.05), respectively, while DNA levels of the comet tail (TailDNA%, TDNA%) were 72.21±14.61%, 23.11±4.98%, and 1.02±1.41% (P<0.05), respectively. The corresponding DNA damage was categorized as high (grade 3), moderate (grade 2), and no damage (grade 0). CONCLUSIONS Different sizes of OMVs induced different degrees of DNA damage in intestinal epithelial Caco-2 cells.
背景 近期研究表明，大肠杆菌引起的消化道疾病可能与外膜囊泡（OMVs）诱导肠上皮细胞中的肠道双链断裂（DSBs）有关。本研究旨在比较OMVs对肠上皮Caco-2细胞中DSBs的影响，并为大肠杆菌引起的消化系统疾病提供新的治疗方法。 材料与方法 制备大肠杆菌OMVs并与Caco-2细胞共培养。通过共聚焦显微镜观察Caco-2细胞对OMVs的摄取。通过蛋白质免疫印迹法检测γ-H2AX蛋白。通过单细胞凝胶电泳检测由OMVs引起的DSBs。 结果 粒度分析仪显示，在20000×g和50000×g离心的OMVs的平均直径分别为217.5±7.29nm和186.3±6.59nm（P<0.05）。OMVs的透射电子显微镜显示具有各种不同大小的脂质双层结构。共聚焦荧光显微镜显示，24小时后OMVs几乎完全进入Caco-2细胞。在20000×g和50000×g离心的OMVs以及对照组（无OMVs）中，γ-H2AX蛋白条带灰度值归一化数据的比例分别为2.23±0.18、1.58±0.20、1±0.30（P<0.05），而彗星尾的DNA水平（TailDNA%，TDNA%）分别为72.21±14.61%、23.11±4.98%和1.02±1.41%（P<0.05）。相应的DNA损伤分为高（3级）、中（2级）和无损伤（0级）。 结论 不同大小的OMVs在肠上皮Caco-2细胞中诱导不同程度的DNA损伤。
