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在延缓性免疫排斥反应(DXR)过程中,NF-kB发挥着关键作用.如何恰到好处地抑制其活性是本领域研究的重要问题之一.用改造的ElA基因(ElA△)包括功能区(1～80氨基酸)和核定位区(139~243氨基酸),删除其中可能对人体有害的CR2区,将其克隆到真核表达载体pcDNA3中,并转染猪血管内皮细胞(PAEC),经G418筛选,获得稳定表达细胞株.RT-PCR技术和细胞生长曲线分析,证明ElA△基因能在PAEC中稳定表达,且不影响细胞的正常生长,并能抵抗肿瘤坏死因子-α(TNF-α)诱导的细胞凋亡.报告基因分析表明,ElA△能抑制由TNF-α诱导的NF-kB活性,其抑制率为53％,对NF-kB信号转导途径下游的一个重要炎症基因——E-选择素基因的表达抑制率达63％.综上,ElA△基因的这些功能基本符合异种器官移植中克服DXR的要求,为利用ElA△基因克服DXR的可行性提供了实验依据.
NF-kB plays a key role in the process of delayed immune rejection (DXR), and how to inhibit the activity of NF-κB is one of the most important problems in the field of research.In this study, ElA △ ~ 80 amino acids) and the nuclear localization region (139 ~ 243 amino acids). The CR2 region, which may be harmful to humans, was deleted and cloned into the eukaryotic expression vector pcDNA3 and transfected into the porcine vascular endothelial cells (PAEC) , Stable expression of cell lines was obtained.RT-PCR and cell growth curve analysis showed that ElA △ gene can be stably expressed in PAEC without affecting the normal growth of cells, and can resist the induction of tumor necrosis factor-α (TNF-α) .Analysis of reporter gene showed that ElA △ could inhibit NF-κB activity induced by TNF-α with a 53% inhibition rate, which is an important inflammatory gene downstream of NF-κB signal transduction pathway, E- In conclusion, these functions of ElA △ gene basically conformed to the requirement of DXR in xenotransplantation, which provided an experimental basis for using the ElA △ gene to overcome the feasibility of DXR.