斑点叉尾(鱼回)呼肠孤病毒诱导斑点叉尾(鱼回)肾脏组织细胞凋亡的研究

摘要

为研究斑点叉尾(鱼回)呼肠孤病毒(CCRV)诱导斑点叉尾(鱼回)肾脏细胞(CCK)发生凋亡的机理,以CCRV感染的CCK细胞为实验材料,采用Hoechst 33258染色、DNA片段化检测、TUNEL反应、亚G1期细胞检测以及线粒体膜电位变化检测等方法进行实验.感染试验结果显示,病毒感染斑点叉尾(鱼回)肾脏组织细胞后,细胞变圆、皱缩,随后细胞脱落,细胞单层呈网状,感染72 h后出现典型细胞病变效应(CPE)；病毒感染48 h后Hoechst 33258染色结果显示,细胞的染色质固缩、核边缘化或破裂,可观察到凋亡小体,细胞凋亡率随时间增加；DNA片段化检测结果显示,病毒感染细胞12 h后细胞基因组DNA出现片段化,随后逐渐增强,72 h达到最高；TUNEL反应结果表明,病毒感染细胞72 h后细胞基因组DNA断裂,有大量游离3’末端自由羟基(-OH)存在.亚G1期细胞检测结果显示,病毒感染48 h后,约53.44％细胞处于亚G1期；利用JC-1检测试剂盒检测病毒感染细胞的线粒体膜电位变化,病毒感染细胞24 h后线粒体膜通透性发生改变,膜电位变化显著.紫外线灭活与热灭活的斑点叉尾(鱼回)呼肠孤病毒不能诱导斑点叉尾(鱼回)肾脏细胞发生凋亡,表明细胞凋亡依赖于病毒复制.%Channel catfish reovirus(CCRV)is the pathogen of the hemorrhage of channel catfish (Ictalurus punctatus Rafinesque) , which propagates in the kidney of channel catfish ( CCK) cell line. In this study, the evaluation of whether the CCRV could induce the apoptosis of CCK cells was examined by Hoechst33258 staining,DNA fragmentation assay, TUNEL reaction, and flow cytometry analysis using JC-1, etc. The infection test indicated that CCRV caused typical cytopathic effect ( CPE) in CCK cells. Chromatin condensation,nuclei marginalization and the apoptotic bodies were observed in Hoechst 33258 staining,and the apoptotic rate increased with the time going of CCRV infection. The DNA fragmentation assay demonstrated that fragmentation was first noted at 12 h post-infection and reached the peak at 72 h post-infection. In addition,genomic DNA was broken and lots of 3'-termianl free-hydroxyl group( - OH) were generated in infected CCK in TUNEL assay. The hypo-diploid fraction was shown in the sub-G1 cells analysis and the apoptotic rate was 53. 44% at 48 h post-infection. Furthermore, the change of the mitochondrial membrane potential(MMP) was investigated by flow cytometric analysis with JC-1 fluorescent labeling and the result showed that the membrane permeability and MMP of CCK cells changed significantly at 24 h post-infection. These results demonstrated that CCRV induced apoptosis in CCK cells. Besides, CCRV-induced apoptosis did require the viral replication as the apoptosis was blocked in CCK cells by both heat-inactivated and UV-inactivated virus.