抗人TNF单克隆抗体对TNF诱导的NF-κB核转位的抑制作用

The inhibition of TNF-induced NF-κB nuclear translocation in ECV304 cells by mAbs against human TNF

目的 :鉴定 3株抗人TNF单克隆抗体 (mAb)D2、E6和F6对TNF诱导的NF κB核转位的抑制作用。方法 :将不同浓度的 3株mAb及无关mAb分别与TNF溶液孵育后 ,加入到ECV30 4细胞培养液中。孵育 1h后收获细胞 ,从中提取核蛋白并测定其含量 ,用电泳迁移率变动分析 (EMSA)检测核提取液中NF κB的核转位。结果 :3株抗TNFmAb均可抑制TNF诱导的ECV30 4细胞中NF κB的核转位。其中mAbD2的抑制作用最强 ,其次为mAbF6和E6 ,无关对照抗体也有一定的非特异性反应。抑制作用与加入的mAb呈良好的剂量依赖关系 ,在 10mg/L和 0 .1mg/L条件下 ,mAbD2的抑制率分别为 94 .2 %和75 .1% ,mAbE6分别为 6 4 .9%和 2 8.6 % ,mAbF6分别为 70 .3%和 4 9.5 % ,无关对照抗体分别为 2 0 .0 %和 11.1%。结论 :mAbD2、E6和F6可特异性地抑制TNF诱导的NF κB核转位 。

AIM: To identify the inhibition of TNF-induced NF-κB nuclear translocation by three anti-human TNF mAbs, D2, E6 and F6. METHODS: TNF solutions were pretreated with mAbs D2, E6 and F6 as well as control mAb at 37℃ for 1 h, respectively, and then they were added to ECV304 cell cultures. After 1 hour, the cells were harvested and nuclear proteins were extracted. The NF-κB activity in nuclear extract was detected by electrophoretic mobility shift assay (EMSA). RESULTS: All of the three anti-TNF mAbs could inhibit TNF-induced NF-κB nuclear translocation in a dose-dependent manner, but control mAb had also some non-specific reaction. At the concentrations of 10 mg/L and 0.1 mg/L, the inhibition rates of mAb D2, E6 and F6 were 94.2% and 75.1%, 64.9% and 28.6%, 70.3% and 49.5% respectively, while the inhibition rate of control mAb was only 20.0% and 11.1%. CONCLUSION: mAbs D2, E6 and F6 can specifically inhibit TNF-induced NF-κB nuclear translocation, which lays the foundation for preparation of therapeutic chimeric anti-human TNF antibody for freatment of infectious and autoimmune diseases.