Bcl-2與caspase抑制劑調控牛流行熱病毒複製及誘發細胞凋 亡之研究

牛流行熱病毒 (bovine ephemeral fever virus, BEFV)為負向單股 RNA病毒，感染牛、乳牛及水牛等，受感染牛隻會突發性地發高燒、鼻子出現分泌物、跛腳、便秘及泌乳量減少等臨床病徵，影響經濟甚巨。BEFV於2004年被證實具有誘發細胞凋亡能力。本研究主要應用抗凋亡基因 (bcl-2)和抑制凋亡之試劑 (caspase inhibitors)來探討BEFV在細胞內誘發凋亡及複製之機序。可大量表現(over-expression)bcl-2基因之BHK-21細胞株 (stable clone)已由本實驗室建立。藉由DNA fragmentation分析、活化態caspases 8、9及3的螢光染色分析及caspase inhibitors抑制試驗等，證實BEFV誘導BHK-21細胞凋亡乃透過caspase-dependent之機序，而在BHK-21/bcl-2細胞則因抗凋亡之bcl-2蛋白之調控，不論在DNA fragmentation 分析、活化態caspases 9及3的螢光染色及cytochrome c釋放至細胞質之偵測，皆證實大量表現bcl-2基因能延緩病毒誘發細胞凋亡發生。在抑制試驗也證實caspase inhibitors皆可抑制上中下游之caspases8、9及3之活化。Caspase inhibitors 8、9及3可抑制細胞凋亡之百分比分別為58.1、71.0及64.7。本實驗透過使用大量表現bcl-2之細胞株及caspase抑制劑，證實BEFV誘發細胞凋亡透過 caspase-dependent及mitochondrial pathway。

關鍵字

牛流行熱病毒；細胞凋亡； bcl-2蛋白

並列摘要

Bovine ephemeral fever virus (BEFV) is a negative single-stranded RNA virus that causes severe infection in cattle, lactating cows, and water buffalo resulting in major economic losses. The disease is characterized clinically by the sudden onset of fever, nasal discharges, lameness, constipation and low lacation. BEFV induction of apoptosis was confirmed in 2004. In the present study we used bcl-2 and caspase inhibitors to inhibit BEFV induced apoptosis and virus replication. Our laboratory have previously established the bcl-2 stable clones (BHK-21/bcl-2). To assay apoptosis induction levels we did DNA fragment assay, caspases 8, 9 and 3 activation and the effect of caspase inhibitors and bcl-2 on BEFV-induced apoptosis, demonstrating that BEFV-induced apoptosis was caspase-dependent. Our results also demonstrated that BHK-21/bcl-2 can delay apoptosis and inhibit cytochrome c release into cytosol. Inhibition of caspases 8, 9, and 3 demonstrated that caspase inhibitors could inhibit activated caspase 8, 9, and 3 hence apoptosis. The inhibition percent of caspase inhibitors 8, 9, and 3 were 58.1, 71.0, and 64.7 respectively. In conclusion, this study demonstrated that BEFV-induced apoptosis was caspase-dependent and involved the mitochondrial pathway.

並列關鍵字

BEFV ； apoptosis ； bcl-2

參考文獻

林朝舜。牛流行性感冒。家畜衛生。台灣省政府農林廳暨中國農村復興聯合委員會。54-61頁，1973。

Burgess, G. W. (1917). Bovine ephemeral fever: a review. Vet. Bull. 41, 887-895.

Hsu, H. S., Liao, Y. K., Hun, H. H. (1997). The seasonal successional investigation of Culicoides spp. In cattle farms of Pingtung district, Taiwan. J. Chin. Soc. Vet. Sci. 23, 303-310.

Atkinson, E. A., Barry, M., Darmon, A. J., Shostak, I., Turner, P. C., Moyer, R. W., Bleackley, R. C. (1998). Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B. J. Biol. Chem. 273, 21261-21266.

Burgess, G. W., Genoweth, P. J. (1975). Mid-piece abnormalities in bovine semen following experimental and natural cases of bovine ephemeral fever. Br. Vet. J. 131, 536-540.

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Bcl-2與caspase抑制劑調控牛流行熱病毒複製及誘發細胞凋亡之研究

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