非复制型痘苗病毒天坛株NTV诱发早期细胞凋亡

Non-replicating vaccinia virus Tiantan strain NTV induces early apoptosis

目的对非复制型痘苗病毒NTV感染人源细胞后的细胞形态变化及相关分子机制进行研究,为NTV载体的进一步优化改造和应用提供科学依据。方法用复制型痘苗病毒天坛株VTT和非复制型痘苗病毒NTV感染HeLa细胞,观察细胞的形态学变化。然后收获细胞,电泳检测rRNA断裂水平和Western blot检测凋亡相关分子信号,初步确定NTV诱导的早期细胞凋亡的通路与机制。结果在细胞形态学方面观察到被NTV感染的细胞,与VTT相比,能够在较早时间出现细胞变圆、皱缩等病变现象。DAPI染色观察到被NTV感染的细胞核在早期会出现染色质高度凝聚、边缘化的现象并随着时间的增加而崩解。rRNA断裂水平检测结果说明被NTV感染16 h后的rRNA已发生断裂和降解。Western blot检测结果说明在NTV感染HeLa细胞中能够检测到比正常细胞中更强的PARP、caspase-3及caspasee-9的分子信号,但caspasee-8并没有明显增加,而VTT的结果则与NTV相反。结论NTV在早期能够诱导细胞凋亡,为痘苗病毒载体改造提供理论依据。

Objective To study the cell morphological changes and related molecular mechanisms of non-replicating vaccinia virus NTV infection with human cells and to provide a scientific basis for the further optimization,transformation and application of NTV vectors.Methods HeLa cells were infected with vaccinia virus Tiantan strain VTT and non-replicating vaccinia virus NTV,and the morphological changes of cells were observed.Then cells were harvested,and rRNA break levels were detected by electrophoresis and the molecular signals associated with apoptosis were detected by Western blotting,and the pathways and mechanisms of NTV-induced early apoptosis were preliminarily determined.Results In this study,in terms of cell morphology,it was observed that cells infected with NTV were able to have cell rounding,wrinkles and other lesions at an earlier time compared with VTT.DAPI staining showed that NTV-infected nuclei exhibited high chromatin aggregation,marginalization,and disintegration over time.The rRNA fracture level test result indicate that rRNA has been broken and degraded after 16 hours of NTV infection.The Western blotting test result showed that the molecular signals of PARP,caspase-3 and caspase-9 that were stronger than in normal cells could be detected in NTV-infected HeLa cells,but there was no significant increase in caspase-8,while the result of VTT were the opposite of those in NTV.Conclusions NTV can induce apoptosis in the early stage and provide a theoretical basis for the modification of vaccinia vectors.