蓝舌病毒湖北株致人肝癌Hep-3B细胞和人肺腺癌A549细胞死亡机制的研究

Death mode of Hep-3B and A549 tumor cells induced by bluetongue virus strain HbC_3

目的探讨蓝舌病毒湖北株(BTV-HbC3)致人肝癌Hep-3B和人肺腺癌A549细胞死亡的方式及其作用机制。方法观察细胞感染BTV-HbC3的病变效应;原位末端标记(TUNEL)法检测细胞感染BTV-HbC3是否出现凋亡;激光打描共聚焦显微镜检测荧光标记的细胞核及内质网变化;萤光照度仪检测病毒诱导细胞caspase-3/7、caspase-8和caspase-9的活性变化。结果BTV-HbC3感染Hep-3B细胞后有明显的病变效应,大量细胞凋亡,细胞核染色质边聚,内质网大量空泡形成,caspase- 3/7活性升高,caspase-8和caspase-9未升高;BTV-HbC3感染A549细胞后有明显病变效应,仅见零星凋亡细胞,内质网有空泡形成,细胞核未见染色质边聚,caspase-3/7和caspase-9活性升高,caspase-8未升高。结论BTV-HbC3主要通过细胞内内质网途径诱导Hep-3B细胞凋亡而发挥其抑瘤作用,而对人肺腺癌的抑瘤作用则是通过诱导A549细胞的非凋亡性程序性死亡来实现的。

Objective To study the death mode of human hepatocellular carcinoma Hep-3B cells and human lung adenocarcinoma A549 cells induced by bluetongue virus strain HbC3 （BTV-HbC3） and the mechanism of its action. Methods BTV-HbC3 was used to infect the tumor ceils, and the cytopathic effects （CPE） was observed. TUNEL staining was used to detect the apoptosis of tumor cells induced by BTV- HbC3. The changes of endoplasmic reticulum and nuclei treated with BTV-HbC3 were further examined by laser scanning confocal microscopy. The activities of caspase-3/7, caspase-8 and caspase-9 were determined by fluorescence analysis. Results Hep-3 B cells were sensitive to BTV-HbC3. Lots of early apoptotic cells were found by TUNEL staining. The laser scanning confocal microscopic examination showed characteristics of apoptosis, such as pyknotic nuclei, margination of nuclear chromatin and vacuolization of endoplasmic reticulumin in Hep-3B cells exposed to BTV-HbC3. The activity of caspase-3/7 was increased, but the activity changes of caspase-8 and caspase-9 were not found. A549 cells were sensitive to BTV-HbC3 too. But no apoptotic cells were observed by TUNEL staining. The results of laser scanning confocal microscopy showed marked vacuolization of endoplasmic reticulum, but chromatin margination was not found after ASd9 cells was exposed to BTV-HbC3. The activity of caspase-3/7 and caspase-9 was increased, but the activity of caspase-8 was not changed. Conclusion BTV-HbC3 induces apoptosis of Hep-3B tumor cells mainly through endoplasmic reticulum signal transduction pathway, and the features of cell death in A549 cells could be described as paraptosis.