Dai Chaohui, Wang Haifei, Zhu Guoqiang, Wu Shenglong, Bao Wenbin
Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China.
College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, China; Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Jiangsu, Yangzhou 225009, China.
Gene. 2017 Sep 10;628:267-274. doi: 10.1016/j.gene.2017.07.055. Epub 2017 Jul 20.
Cluster of differentiation antigen 14 (CD14) is the membrane receptor protein in Toll-like Receptor 4 (TLR4) signaling pathway, which plays an important regulation role in not only innate immune response but also adaptive immune response. In this study, the pig kidney epithelial cell (PK15) line with CD14 gene silencing mediated by lentivirus was established and cells of CD14-RNAi and NC group were exposed to three kinds of Escherichia coli (E. coli F18ab, E. coli F18ac and E. coli K88ac) and LPS. Then qPCR and western blot were used to detect expression levels of TLR4 signaling pathway-related genes. Finally, ELISA was used to detect the level of proinflammatory cytokines in the cell culture supernatant. The results showed that the expression level of TLR4 signaling pathway-related genes in the entire signal pathway had obvious increases when cells were exposed to the stimulation induced by E. coli and LPS. In addition, the expression levels of CD14-RNAi group were overall significantly lower than NC group (P<0.05 or P<0.01), which was the same with the release levels of proinflammatory cytokines. This study revealed that pig CD14 gene silencing partially inhibited immune response to E. coli F18 invasion mediated by TLR4 signaling pathway.
分化簇抗原14（CD14）是Toll样受体4（TLR4）信号通路中的膜受体蛋白，其不仅在天然免疫应答中，而且在适应性免疫应答中均发挥重要的调节作用。本研究构建了慢病毒介导的CD14基因沉默的猪肾上皮细胞（PK15）系，并将CD14-RNAi组和NC组细胞分别暴露于三种大肠杆菌（大肠杆菌F18ab、大肠杆菌F18ac和大肠杆菌K88ac）及脂多糖（LPS）中。随后采用qPCR和蛋白质免疫印迹法检测TLR4信号通路相关基因的表达水平。最后，采用酶联免疫吸附测定法（ELISA）检测细胞培养上清中促炎细胞因子的水平。结果显示，当细胞受到大肠杆菌和LPS诱导的刺激时，整个信号通路中TLR4信号通路相关基因的表达水平均显著升高。此外，CD14-RNAi组的表达水平总体上显著低于NC组（P<0.05或P<0.01），促炎细胞因子的释放水平亦是如此。本研究表明，猪CD14基因沉默可部分抑制由TLR4信号通路介导的对大肠杆菌F18侵袭的免疫应答。
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