洪伟,饶凤琴,吴昌学,赵行行,程玉梅,张婷,齐晓岚,禹文峰,官志忠
HONG Wei,RAO Fengqin,WU Changxue,ZHAO Xingxing,CHEN Yumei,ZHANG Ting,QI Xiaolan,YU Wenfeng,GUAN Zhizhong

• 1:贵州医科大学地方病与少数民族疾病教育部重点实验室&贵州省分子生物学重点实验室
• 2:贵州医科大学附院ICU

摘要(Abstract)：

目的:研究人肠道来源艰难梭感染Caco-2细胞对NF-κB信号通路相关蛋白表达的影响。方法:用艰难梭菌毒素A和毒素B感染Caco-2细胞24 h,以未感染艰难梭菌毒素A和毒素B的Caco-2细胞为对照组,蛋白质免疫印迹法检测真核细胞核因子(NF-κB)通路蛋白P-p65和p65的表达; Caco-2细胞用6孔板制作爬片,待细胞长满6孔板后,以感染复数(MOI)=100加入艰难梭菌,与Caco-2细胞共培养3、6、9、12及24 h,革兰染色后油镜观察,记录黏附细菌数与细胞数,计算共培养时间与细菌黏附数量之间的关系;艰难梭菌感染Caco-2细胞12 h,以Caco-2为对照组,蛋白质免疫印迹法检测NF-κB通路蛋白P-p65、p65、IKKα及IκBα的表达。结果:艰难梭菌与Caco-2细胞共培养12 h时黏附数最多,P-p65和IKKα蛋白表达增加(P<0.05),p65和IκBα蛋白表达降低,差异有统计学意义(P<0.05)。结论:人源艰难梭菌感染Caco-2细胞可激活NF-κB信号通路。
Objective: To study the effect of human intestinal tract-derived Clostridioides difficile mediated-infection on expression of NF-κB signaling pathway-related proteins in Caco-2 cells. Methods: Caco-2 cells were infected with C. difficile toxin A and toxin B for 24 h. Uninfected Caco-2 cells were used as a control group. Western blot was used to detect NF-κB pathway-related proteins such as p65, p65. In addition, Caco-2 cells grown full on 6-well plate were co-cultured with C. difficile with MOI 100 for 3, 6, 9, 12 and 24 h, stained with Gram staining kit and observed under oil microscope. The number of adhered bacteria and the number of cells were recorded. The relationship between co-cultivation time and the number of adhered bacteria was analyzed. At 12 h post-infection with C. difficile, the expression levels of phospho-p65, p65, IKKα and IκBα were measured by Western blot. Results: The adherence of C. difficile on Caco-2 cells reached the maximum at 12 h after-infection. The expression levels of phospho-p65 and IKKα proteins were significantly increased(P<0.05), while the expression of p65 and IκBα proteins were significantly decreased(P<0.05). Conclusion: C. difficile infection activates NF-κB signaling pathway in Caco-2 cells.

关键词(KeyWords)： 人源艰难梭菌;肠上皮细胞;炎症;凋亡;NF-κB信号通路
C.difficile;intestinal epithelial cells;inflammation;apoptosis;NF-κB signaling pathway

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31560318,31601012,31760318);; 贵州省自然科学基金资助项目[黔科合基础(2019)1441,(2018)1132,(2018)5779-17]

作者(Author): 洪伟,饶凤琴,吴昌学,赵行行,程玉梅,张婷,齐晓岚,禹文峰,官志忠
HONG Wei,RAO Fengqin,WU Changxue,ZHAO Xingxing,CHEN Yumei,ZHANG Ting,QI Xiaolan,YU Wenfeng,GUAN Zhizhong

DOI: 10.19367/j.cnki.1000-2707.2019.12.001

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