肿瘤坏死因子α-863C／A多态性影响核因子-κB结合活力在脓毒症发病中的作用及机制

The effect of single nucleotide polymorphism of -863C/A of TNF-α on binding activity of nuclear factor kappa B with DNA and the mechanism involved in sepsis

目的探讨TNF—α．863C／A基因多态性对NF—gBp65．p50和p50．p50活化的调控，以及在脓毒症发病中的作用及机制。方法选择已知TNF-α-863C／A基因多态性的脓毒症患者30例，采集静脉血，用LPS刺激后，用ELISA法测定上清中TNF—α表达；用EMSA方法测定NF-κBp65-p50和p50-p50活化水平。结果外周血经LPS刺激后NF—κB活化和TNF-α表达存在时相性变化，1-2周明显低于0及第4周水平；具有-863A等位基因的患者其NF—gB活化模式以p50-p50为主，明显高于C／C组水平，p65-p50：p50-p50的比值明显低于C／C组（0．4±0．2）比（0．7±0．4），相应地TNF-α表达随NF-κB两种二聚体比值的降低而明显下降。结论不同的TNF- α-863C／A基因型能调节NF—κBp65-p50和p50-p50二聚体活化后与DNA结合水平进而介导下游TNF-a表达的改变。

Objective To investigate the effect of -863C/A polymorphism of TNF-α gene on the NF-kappa B binding activity with DNA motif, and the regulation of TNF-α expression involved in the mechanism of sepsis. Methods Thirty patients with sepsis were divided into three groups according to the gene type of -863 C/C, C/A, A/A. The blood sample was harvested and stimulated with LPS. The culture supernatant TNF-α production was measured by ELISA, and the activation of p65-p50 or p50-p50 in WBC nuclei was examined by EMSA. Results In vitro,the activation of NF-kappa B and expression of TNF-α in the whole blood stimulated with LPS were significantly lower at 1st and 2nd week after sepsis than at 0 and 4th week of sepsis. The activation style of NF-kappa B in genotypes with -863A characterized with p50-p50 predominant and the level of p50-p50 in nuelel was higher than that of C/C genotype group. In contrast ,the ratio of p65-p50 to pS0-pS0 was lower in C/A and A/A groups than in C/C group （0.4 ± 0. 2 vs 0.7 ± 0.4 ）. Meanwhile ,the expression of TNF-α was decreased significantly along with the lower acti- vation of p65-p50 dimer. Conclusion The data indicated that different genotypes of 863C/A polymorphism on TNF-α gene could regulate NF-kappa B p65-p50 and p50-p50 activation and the binding with DNA motif and the expression of downstream gene including TNF-α.