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为研究斑点叉尾呼肠孤病毒(CCRV)诱导斑点叉尾肾脏细胞(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72h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呼肠孤病毒不能诱导斑点叉尾肾脏细胞发生凋亡,表明细胞凋亡依赖于病毒复制。
To investigate the mechanism of CCRV-induced apoptosis in CCK cells, CCRV-infected CCK cells were used as experimental materials. Hoechst 33258 staining, DNA fragmentation assay, TUNEL Reaction, sub-G1 cell detection and mitochondrial membrane potential changes detection methods such as experiments. The results of infection showed that the cells became rounded and collapsed after infected with kidney cells of Ictalurus punctatus, then the cells became exfoliated and the monolayer became reticular. The typical cytopathic effect (CPE) appeared after 72 hours of infection. Post-Hoechst 33258 staining showed that apoptotic bodies could be observed in apoptotic bodies after chromatin condensation, nucleus margination or rupture. DNA fragmentation assay showed that the cells infected with virus at 12 h Genomic DNA was fragmented and then increased gradually and reached the highest level at 72 h. The TUNEL assay showed that the genomic DNA was broken after 72 h in virus-infected cells, and a large number of free hydroxyl groups (-OH) were found at the 3 ’end. The results of sub-G1 phase assay showed that 53.44% of cells were in sub-G1 phase 48 h after virus infection. The mitochondrial membrane potential of virus-infected cells was detected by JC-1 kit. The mitochondrial membrane permeability Sexual changes, membrane potential changes significantly. UV-inactivated and heat-inactivated channel catfish Reovirus can not induce apoptosis in the channel catfish, suggesting that apoptosis is dependent on viral replication.