【摘要】 人类巨细胞病毒(HCMV)流行广泛,危害严重,由于其严格的物种特异性限制,动物模型缺乏,严重阻碍了对其致病机制的研究及药物、疫苗的研发。本研究对树鼩(Tupaia belangeri)来源的原代真皮成纤维细胞感染HCMV后的细胞死亡现象进行了初步探索。首先,观察了感染后细胞病变和死亡情况,使用细胞增殖检测试剂盒测定了细胞活力;其次,对凋亡相关因子bax、bcl-2和未折叠蛋白反应相关因子chop、atf4、xbp1s转录水平的差异进行了逆转录荧光定量PCR检测与分析;然后利用免疫蛋白印迹技术检测并分析了主要病毒蛋白IE1、UL44和凋亡相关因子Bax、Caspase-9、Caspase-3、PARP的表达水平;最后结合膜联蛋白-V和碘化丙啶双染色法从生物化学角度对细胞死亡类型进行了鉴定。结果显示,细胞病变和死亡情况随着感染进程逐渐严重,细胞活力下降明显(P <0.001)。感染上调bax、bcl-2、chop、atf4、xbp1s转录水平并且使bax与bcl-2转录水平呈明显拮抗趋势;感染同时上调Bax、Caspase-9、Caspase-3、PARP蛋白表达和逐级剪切激活;病毒蛋白IE1、UL44可以上调至一个完整的病毒复制周期结束。综上,本研究指出HCMV跨物种感染树鼩原代真皮成纤维细胞可诱导细胞凋亡,并且与内质网和线粒体密切相关。更多还原

【Abstract】 Human cytomegalovirus(HCMV) is popularly prevalent and extremely harmful, which has seriously hindered the researches on its pathogenic mechanism, drugs and vaccines, owing to restriction of species specificity and lacking animal models. In this study, we have explored the death of primary dermal fibroblasts(TSDF) isolated from the Chinese tree shrew(Tupaia belangeri) that infected with HCMV-Towne.The cytopathic effect and cellular death after infection were observed, and cell viability was measured using CCK(cell counting kit). The differences in transcriptional levels of apoptosis-related factor genes bax, bcl-2,and an unfolded protein response(UPR) relevant factor genes chop, atf4, xbp1 s were detected using qRT-PCR.Western blot was also used to detect principal viral proteins IE1 and UL44 as well as apoptosis-related factors including Bax, Caspase-9, Caspase-3, and PARP. Additionally, apoptosis was detected by AV-PI double staining. The results showed that the cytopathic effect and cellular death gradually worsen as the infection progressed(Fig.1 a-e), accompanying with significant decrease of cell viability(Fig. 2 c, P < 0.001).Moreover infection caused up-regulation of the transcription levels of bax, bcl-2, chop, atf4, xbp1 s, and the bax and bcl-2 transcription levels showed a significant antagonistic trend(Fig. 3). Meanwhile, protein expression and progressive activation of Bax, Caspase-9, Caspase-3, and PARP were also up-regulated.Nevertheless viral proteins IE1 and UL44 were up-regulatied to the end of a complete virus replication cycle(Fig. 4). In summary, our study indicates that HCMV can induce apoptosis through cross-species infecting the tree shrew primary dermal fibroblasts, which is closely related to endoplasmic reticulum and mitochondria.更多还原