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- 2018
LBP基因沉默对LPS诱导水牛单核巨噬细胞炎症相关基因表达的影响
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Abstract:
为了探讨抑制脂多糖结合蛋白（LBP）基因表达对水牛单核巨噬细胞在内毒素（LPS）诱导下炎症相关基因的表达，首先采用三质粒慢病毒包装系统包装LBP基因shRNA重组质粒pSicoR-GFP-shLBP774.利用病毒颗粒感染水牛单核巨噬细胞，进行荧光定量qRT-PCR及ELISA检测.结果显示，单核巨噬细胞在用5×108 IU/mL滴度的LBP-shRNA774慢病毒颗粒，感染指数（MOI）为300、感染7 d的条件下，感染效率超过50%.qRT-PCR检测结果显示，与LPS对照组相比，shLBP774感染组可极显著抑制LBP基因的表达（p < 0.01），CD14，TLR4，TNF-α，IL-1β，IL-8等基因表达显著降低（p < 0.05）.ELISA检测结果发现，与对照组相比，细胞分泌的TNF-α，IL-1β蛋白量显著降低（p < 0.05）.以上结果表明，抑制LBP基因表达可以有效降低LPS诱导下单核巨噬细胞炎症相关基因的表达，进而调控LPS引发的炎症反应，提示可将LBP作为靶基因深入研究水牛单核巨噬细胞免疫功能和LPS致炎信号传导分子机制.
In order to investigate the effect of inhibition of LBP (lipopolysaccharide binding protein) gene expression on LPS-induced expression of macrophage inflammatory genes in buffalo mononuclear macrophages, LBP-shRNA 774 recombinant plasmid was used to package the virus, and the suitable conditions for target cell infection were explored. Theninflammation-related genes in virus-infected cells were detected with the qRT-PCR and ELISA methods. The results showed that the virus titer of LBP-shRNA774 was > 5×108 IU/mL, MOI was 300, and after 7 days, the infection rate was more than 50%. qRT-PCR showed that compared with the LPS control group, the mRNA level of LBP gene was reduced by 50%, meanwhile CD14, TLR4, TNF-α, IL-1β and IL-8 were decreased significantly (p < 0.05). ELISA demonstrated that the contents of cytokine TNF-α and of IL-1β in the LBP-shRNA774 group were significantly lower than in the LPS control group (p < 0.05). Also, the content of soluble LBP (s LBP) protein was significantly lower (p < 0.05), only accounting for 48.54%. The above results suggested that inhibition of LBP gene expression can effectively reduce the expression of LPS-induced inflammation-related genes of monocyte-macrophage, and regulate the LPS-induced inflammatory response
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