硫喷妥钠预先给药对肿瘤坏死因子-α激活Jurkat细胞NF-κB活性的影响

Effects of thiopental pretreatment on NF-κB activity in TNF-α activated Jurkat cells

目的探讨硫喷妥钠预先给药对肿瘤坏死因子-α(TNF-α)激活Jurkat细胞NF-κB水平的影响。方法选择人T淋巴瘤细胞系Jurkat细胞,浓度调节至1×105/ml,实验分两部分,1．将细胞随机分为6组:空白对照组不加入药物;TNF-α5 U/ml组;400、1 000μg/ml硫喷妥钠组;400μg/ml硫喷妥钠 +TNF-α 5 U/ml组和1000 μg/ml硫喷妥钠+TNF-α 5 U/ml组:加入硫喷妥钠1 h后洗去,再加入5 U/ml TNF-α;在加入TNF-α后1 h提取细胞核蛋白,采用凝胶电泳迁移率分析方法测定NF-κB活性,Western 杂交法对NF-κB的抑制因子IκB-α进行半定量研究。2．将细胞随机分为8组,空白对照组不加入药物;TNF-α5U/ml组;其他6组先用1 000μg/ml硫喷妥钠孵育1 h,随后用PBS冲洗,加入新鲜完全培养基,分别在培养后即刻、2、4、6、8、10 h加入TNF-α 5 U/ml刺激1 h,用上述方法测定NF-κB活性。结果 TNF-α可激活Jurkat细胞的NF-κB活性,下调IκB-α表达,1000μg/ml硫喷妥钠可抑制TNF-α激活 Jurkat细胞的上述改变。1000μg/ml硫喷妥钠被洗脱后8 h以内对NF-κB活性有抑制作用。结论 1 000μg/ml硫喷妥钠对人淋巴瘤Jurkat细胞TNF-α激活的NF-κB活性具有一定的抑制作用,与上调 IκB-α表达有关,而且可以被该细胞记忆数小时。

Objective To investigate the effects of thiopental （TP） pretreatment on nuclear factor-κB （NF-κB） activity in TNF-α activated Jurkat cells. Methods Jurkat cells of a human T lymphoma cell llne （from incubate preservation center, Wuhan University） were used. The Jurkat cells were incubated in 5% CO2 incubator at a concentration of 1 × 10^5 ·ml^-1 at 37℃ for 24h. The experiment was performed in 2 parts. In part A the cells were divided into 6 groups： group A1 blank control; group A2 TNF-α 5 U· ml^-1 ; group A3 TP 400 μg·ml^-1 ; group A4 TP 1 000 μg· ml^-1 ; group A5 TP 400 μg· ml^-1 ＋ TNF-α 5 U·ml^-1 and group A6 TP 1 000 μg·ml^-1 ＋ TNF-α 5 U · ml^-1 . In group A5 and A6 the cells were incubated first in the presence of rip 400 or 1 000 μg· ml^-1 for 1 h. The cells were then washed and incubated in the medium containing TNF-α 5 U· ml^-1 for lh. The nuclear protein of the cells was then collected for determination of NF-κB activity using electrophoretic mobility shift assay （EMSA） and expression IκB-α protein （an inhibitory protein of NF-κB） using Western blotting. In part B the cells were divided into 8 groups： group B1 blank control; group B2 TNF-α 5 U·ml^-1 ; group B 3-8 in which the cells were incubated in the presence of rip 1 000 μg· ml^-1 for 1 h and then washed and incubated in the presence of TNF-α 5 U·ml^-1 for 1 h immediately （B3） or after 2, 4, 6, 7, 8 h （B4-8）. The cells were then collected for determination of NF-κB activity. Results The NF-κB activity was significantly increased and IκB-α expression significantly downregulated in the TNF-αactivated Jurkat cells as compared with the blank control. The increase in NF-κB activity and down- regulation of IκB-α protein expression in the Jurkat cells induced by TNF-α could be inhibited by pretreatment with thiopental 1 000 μg· ml^-1 . The inhibitory effect of thiopental pretreatment persisted up to 8 hours after being washed off. Conclusion Thiopental pretreatment can inhibit the increase in NF-κB activity in Jurkat cells induced by TNF-α. The inhibitory effect of thiopental pretreatment may be related to up-regulation of IκB-α expression and persists up to 8 hours.