猪繁殖与呼吸综合征病毒诱导的缺失第3外显子的肿瘤坏死因子受体介导细胞凋亡能力的探究

Apoptosis Ability of an Exon 3-skipped Tumor Necrosis Factor Receptor Induced by Porcine Reproductive and Respiratory Syndrome Virus

肿瘤坏死因子受体(TNFR)可以在肿瘤坏死因子(TNF)超家族成员的作用下引起受体介导的细胞凋亡。为了探究猪繁殖与呼吸综合征病毒(PRRSV)和TNFR介导的细胞凋亡的关系,首先使用GenBank中发表的猪源TNFR1的序列设计并合成针对TNFR1的特异性引物,然后检测PRRSV感染猪肺泡巨噬细胞(PAM)后TNFR1的表达情况。结果表明,在PRRSV感染的PAM细胞中,有两种分子量不同的TNFR1存在,序列测定分析表明,一种为野生型TNFR1,另一种为第3外显子缺失(129 bp)的TNFR1(TNFR1-d3)。为探究缺失型TNFR1-d3在细胞凋亡中的意义,我们采用过表达的方法将携带TNFR1和TNFR1-d3的真核表达载体分别转染于HEK293细胞中,用TNF-α结合放线菌酮共刺激细胞诱导凋亡。通过流式细胞术检测发现,与野生型TNFR1相比,TNFR1-d3不再具有促进细胞凋亡的功能,说明缺失的第3外显子参与调控TNF-α诱导的细胞凋亡。本研究阐明了PRRSV和细胞凋亡的新机制。

Tumor necrosis factor receptor(TNFR) can induce receptor-mediated apoptosis under the action of tumor necrosis factor(TNF) superfamily members. To investigate the relationship between porcine reproductive and respiratory syndrome virus(PRRSV) and TNFR-mediated apoptosis, we first designed and synthesized specific primers for TNFR1 according to the sequence published in GenBank, and then detected the expression of TNFR1 in porcine alveolar macrophages at 24 hours post PRRSV infection. We found that there were two different forms of TNFR1. Based on the sequence analysis, one was the wild-type TNFR1, and the other one was a TNFR1 spliced variant with the deletion of exon 3(129 bp). In order to explore the function of TNFR1-d3 in apoptosis, HEK293 cells were transfected with eukaryotic expression vectors carrying TNFR1 or TNFR1-d3, and cell apotosis was induced by TNFα combined with cycloheximide. The flow cytometry data showed that TNFR1-d3 lost its ability to induce apoptosis, suggesting that exon 3 of TNFR1 is involved in TNFα-induced apoptosis. This study elucidates the novel relationship between PRRSV and apoptosis.