猪传染性胃肠炎病毒诱导ST细胞凋亡的初步研究

Preliminary study on apoptosis of ST cells induced by TGEV

【目的】探讨猪传染性胃肠炎病毒(TGEV)感染猪睾丸细胞(ST细胞)后是否可诱导细胞发生凋亡,并对凋亡的信号通路进行初步探究,为进一步揭示TGEV的致病机制提供理论基础。【方法】用TGEV感染对数生长期的ST细胞,同时设立对照组,在感染后不同时间取细胞样品,通过细胞形态观察、细胞活性检测、细胞凋亡率检测和细胞内Caspase-3、Caspase-8、Caspase-9活性检测及Western blot分析等方法,研究TGEV感染对ST细胞的影响。【结果】TGEV感染可抑制ST细胞生长,并且呈现病毒感染剂量和感染时间的依赖性。倒置显微镜和荧光显微镜观察结果显示,10MOI(感染复数)的TGEV感染24h后,ST细胞出现典型的凋亡特征,细胞皱缩变圆、聚堆,细胞核内染色质固缩。流式细胞仪检测及Caspases活性检测表明,感染TGEV的ST细胞凋亡比例随感染时间的延长而逐渐增加,细胞内Caspase-3、Caspase-8和Caspase-9活性逐渐升高。Western blot分析表明,Caspase-3、Caspase-8和Caspase-9无活性的酶原形式随着TGEV感染时间的延长逐渐降解,同时Caspase-3、Caspase-8和Caspase-9的活化片段逐渐增加。TGEV感染能够促进FasL和Bax的表达而下调Bcl-2的表达。【结论】TGEV感染能够诱导ST细胞凋亡,Caspase-3、Caspase-8和Caspase-9及FasL介导的死亡受体通路和Bcl-2家族调控的线粒体凋亡通路,在调控TGEV感染诱导的细胞凋亡过程中可能起着非常重要的作用。

【Objective】This study investigated the effect of TGEV infection on apoptosis of ST cells to improve the understanding on the molecular mechanisms of TGEV-induced cytopathic effect （CPE）.【Method】Sub confluent ST cells were infected or mock infected with TGEV.Infected ST cells and control group cells were collected and analyzed at various times post infection.MTT assay,AO/EB staining,flow cytometry analysis,Caspases activity assay and Western blot assay were used to observe cell morphological changes and determine cell viability,cell apoptotic rate,the activities of Caspase-3,Caspase-8 and Caspase-9,and apoptosis-associated molecules expression.【Result】TGEV-infected ST cells showed decreasing cell viability.Observation with inverted microscope and fluorescent microscope showed that TGEV-infected ST cells had typical apoptotic features,including chromatin condensation and nuclear fragmentation.Flow cytometry analysis of apoptosis showed that the number of apoptotic cells increased gradually over time.Caspases activity assay and Western blot analysis demonstrated that TGEV infection increased the activation of Caspase-3,Caspase-8 and Caspase-9.In addition,Western blot analysis showed that TGEV infection up-regulated the protein levels of FasL and Bax,whereas Bcl-2 was down-regulated.【Conclusion】TGEV induced apoptosis of ST cells,and activations of Caspase-3,Caspase-8,and Caspase-9,FasL-mediated death receptor pathway and mitochondrial-mediated pathway played significant roles.