TM-TNF-α介导的反向信号下调sTNF-α激活后U937细胞因子mRNA的表达

Reverse signaling via TM-TNF-α down-regulated the expression of cytokine mRNA in pre-activated U937 cells by sTNF-α

目的:观测TM-TNF-α介导的反向信号对sTNF-α激活后U937细胞因子mRNA的影响,构建TM-TNF-α胞浆段缺失突变体,并进一步验证。方法:sTNF-α激活U937细胞后撤除,加入可溶性TNFR(sTNFRⅠ)激活TM-TNF-α介导的反向信号,用RT-PCR方法检测反向信号对活化后U937细胞因子IL-1β、IL-8mRNA的影响;采用分子克隆技术构建TM-TNF-α胞浆段缺失突变重组体,采用电穿孔法转染U937,并用Western blot检测蛋白表达。结果:sTNF-α激活U937细胞后撤除,高表达的细胞因子IL-1β、IL-8mRNA随着时间的延长而逐渐降低;sTNFRⅠ激活的TM-TNF-α介导的反向信号可加速细胞因子mRNA的降解,且使mRNA降解至50%的时间分别由约75、60分钟缩短至约45、35分钟。成功构建TM-TNF-α胞浆段缺失突变重组体pcDNA3.0-ΔcsTM-TNF-α并瞬时转染U937细胞,Western blot结果显示U937细胞膜表面除表达26000的野生型TM-TNF-α蛋白外,还可表达约20000的突变体蛋白。这种缺失胞浆段的突变体蛋白可竞争性抑制反向信号对活化后U937细胞因子mRNA的下调作用。结论:TM-TNF-α介导的反向信号可下调sTNF-α激活后U937细胞因子mRNA的表达;且TM-TNF-α胞浆段为其介导的反向信号所必需。

Objective： To investigate the effect of reverse signaling via TM-TNF-α on the expression of cytokine mRNA in preactivated U937 cells by sTNF-α. Besides, the plasmid containing the deleted cytoplasm segment TM-TNF-α mutant gene（ AcsTM-TNF- α） was constructed and transfected into U937 cells so as to confirm this effect. Methods：U937 cells were activated by sTNF-α, then washed and continuously cultured by addition of sTNFR Ⅰ to induce the reverse signal via TM-TNF. The changes of IL-1β and IL-8 mRNA were detected by RT-PCR. The plasmid containing △csTM-TNF-α mutant gene was constructed by molecular clone technique and transiently transfected into U937 cells by electroporation. The expression of TM-TNF-α mutant was assayed by using Western blot. Results ： The high levels of IL-1β and IL-8 mRNA induced by sTNF-α in U937 ceils were found to decline gradually with the prolongation of the time after withdrawing sTNF-α from the culture. Reverse signaling via TM-TNF-α induced by sTNFR I could accelerate the degradation of cytokine mRNA and the half life of IL-1β and IL-8 mRNA reduced from 75,60 min to 45,35 min respectively. The recombinant plasmid pcDNA3.0-△csTM-TNF-α was successfully constructed and transiently transfected into U937 cells. Western blot confirmed that the transfected U937 ceils expressed not only the 26 000 wild type protein produced by itself but also the 20 000 TM- TNF-α mutant. This mutant can competitively inhibit the down-regulation of reverse signaling to cytokine mRNA in pre-activated U937 by sTNF-α. Conclusion： Reverse signaling via TM-TNF-α down-regulated the expression of cytokine mRNA in pre-activated U937 cells by sTNF-α. Furthermore,the cytoplasmic segment of TM-TNF-α is needed for reverse signaling.