Studies on the Mechanism of Avian Reovirus Induced Apoptosis

• Main Authors: Ya-Chun Lee, 李雅純
• Other Authors: Hung-Jen Liu
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/30861983226147309367

碩士 === 國立屏東科技大學 === 生物科技研究所 === 93 === Abstract Student ID: M 9218018 Title of thesis: Studies on the Mechanism of Avian Reovirus Induced Apoptosis Total page: 61 page Name of institute: Graduate Institute of Biotechnology National Pingtung University of Science and Technology Graduate date: 4, July, 2005 Degree conferred: Master thesis Name of student: Ya-Chun Lee Adviser: Hung-Jen Liu, Ph. D. The contents of abstract in this thesis: Avian reovirus（ARV）belongs to orthoreovirus of reoviridae and causes viral arthritis and malabsorption syndrome in chickens. ARV-infected BHK- 21 cells showed a typical pattern of DNA ladder on an agarose gel , demonstrating ARV is able to cause apoptosis in BHK-21cells. To elucidate whether ARV-induced caspase-dependent apoptosis, the activated caspases in ARV-infected BHK-21 cells were examined by caspase ELISA assay. The ELISA assay detected several activated caspases 3, 4, 6, and 9 in ARV-infected BHK-21 cells. When western blot was applied to detect activated caspase 8, Bcl-2, caspase 9, caspase 7, caspase 3, and Poly ADP-ribose polymerase (RARP) in ARV-infected BHK-21 cells, activated caspase 3, caspase 9, and cleaved PARP were detected. Cellular fluorescence could be observed when using caspase special fluorescence dye was used to stain activated caspases 3 and 9 in ARV-infected BHK-21 cells. Caspase inhibitor Z-VAD-fmk（N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone）is able to inhibit the formation of DNA ladder in ARV-infected BHK-21 cells, demonstrating that ARV induces caspase-dependent apoptosis . We also demonstrated that cytochrome c could be released from mitochondria into cytoplasm. Caspase 9 could be activated and then activate the downstream effector caspase 3. The PARP was cleaved by activated caspase 3. ARV σC protein has been demonstrated to be an apoptin by our laboratory. To further study whether σC protein can induce apoptosis by externally added σC into medium, the protein was expressed by baculovirus expression system. Our results suggest that externally added σC into medium can not induce apoptosis in BHK-21 and HeLa cells. The study provided evidences demonstrating that ARV-induced apoptosis requires the activation of internal mitochondrial pathway and induces caspase-dependent apoptosis in BHK-21 cells. Key words: avian reovirus, apoptosis, Western blot