锌指蛋白A20干预人单核细胞炎症因子TNF-α、IL-12以及IL-10表达的实验研究

Interference of gene transfection of A20 zinc finger protein on expression of TNF-α、IL-12 and IL-10 in human monocytes

目的通过阳离子脂质体将A20基因转染入单核细胞,观察A20过表达对促炎因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-12及抗炎因子IL-10表达的调节以及对促炎因子/抗炎因子比率即TNF-α/IL-10和IL-12/IL-10比率的影响,探讨锌脂蛋白A20对单核细胞炎症反应的保护作用及可能的调节机制。方法用Ficoll细胞分离液分离人外周血单核细胞,随机分为A组(空白对照组);B组[脂多糖(lipopolysaccharide,LPS)组];C组(A20转染组);D组(LPS加A20转染组)。荧光显微镜检测GFP报告基因,反转录聚合酶链反应检测内源性A20、外源性A20的mRNA表达;免疫组化检测A20蛋白的表达;双抗体夹心酶联免疫吸附测定(ELISA)方法检测上清液TNF-α、IL-12及IL-10表达水平。结果单核细胞受到LPS(1 mg/L)刺激后,其内源性A20的mRNA和蛋白表达以及TNF-α、IL-12和IL-10表达较对照组均明显升高,差异有统计学意义(P<0.05);TNF-α/IL-10和IL-12/IL-10的比率均明显高于对照组,差异有统计学意义(P<0.05)。转染A20基因的单核细胞,在无LPS刺激的条件下,其内源性A20的mRNA和蛋白表达以及TNF-α、IL-12和IL-10的表达与对照组比较,差异均无统计意义(P>0.05);TNF-α/IL-10和IL-12/IL-10的比率与对照组比较,差异也无统计意义(P>0.05)。而转染A20基因的单核细胞在受到LPS(1 mg/L)刺激后,其促炎因子TNF-α、IL-12的表达均显著低于LPS组,而抗炎因子IL-10的表达明显上调,高于对照组和LPS组;而TNF-α/IL-10和IL-12/IL-10的比率明显下降,低于LPS组,差异有统计学意义(P<0.05)。结论 A20的表达具有炎症活化依赖性;A20过表达可抑制TNF-α、IL-12的表达,而上调抗炎因子IL-10表达,并通过改善促炎因子/抗炎因子的平衡关系,从而达到抑制炎症反应的作用。

Objectives To observe effect of A20 expression on regulating the expressions of proinflammatory cytokine tumor necrosis factor-α （TNF-α） and interleukin （IL）-12, as well as its effect on the ratios of TNF-α IL-10 and IL- 12/IL-10 via transfeeting A20 gene into human monoeytes by cationic liposome, and to discuss the protective function of A20 zinc finger protein on monoeyte inflammatory and potential regulation mechanism. Methods Ficol[ separation medium was used to separate monocytes from peripheral blood. Monocytes were divided randomly into four groups： A group （control group）; B group [lipopolysaccharide （LPS） intervention group]; C group （A20 group） and D group （LPS and A20 intervention group）. GFP gene was detected by fluorescence microscopy. Immunofluorescence and immunohistochemistry were employed to analyze the expression of A20 zinc finger protein. Reverse transcription polymerase chain reaction （RT-PCR） was used to detect A20 mRNA expression. Expression levels of supernatant TNF-α, IL-12 and IL-10 were detected by double antibody sandwich enzyme linked immunosorbent assay （ELISA）. Results The mRNA and protein expressions of endogenous A20, TNF-α, IL-12 and IL-10 increased obviously after monocytes being stimulated by LPS （1 mg/L）;ratios of TNF-α/IL-10 and IL-12/IL-10 were significantly higher than those in control group （P〈0.05）. Comparing with control group, transfection of A20 gene into monocytes, mRNA and protein expressions of endogenous A20,TNF-α,IL-12 and IL-10 had no significant change in the absence of LPS stimulus conditions ; ratios of TNF-α/IL- 10 and IL- 12/IL- 10 had no significant change either （P〉0.05）. However, expressions of TNF-α and IL-12 were significantly lower in LPS and A20 intervention group than those in LPS group,whereas expression of IL-10 increased significantly,higher than those in control group and LPS group after monocytes of transfection A20 gene were stimulated by LPS （1 rag/L）; ratios of TNF-α/IL-10 and 1L-12/IL-10 significantly decreased, which was lower than those in LPS group （P〈0.05）. Conclusions Expression of A20 has inflammatory activation dependency. Over-expression of A20 can inhibit expression of TNF-α and IL-12 and raise expression of inflammatory factor IL-10,thus inhibits the effect of inflammatory response by improving the balance between proinflammatory cytokine and anti-inflammatory cytokine.