周宜黎传染性胰脏坏死病毒凋亡基因vp3.docVIP

周宜黎：傳染性胰臟壞死病毒凋亡基因vp3 所誘發細胞凋亡分子機制之研究 中文摘要 VP3會導致細胞快速凋亡。因此本論文就計劃針對VP3基因功能作進一步探討，想瞭解傳染性胰臟壞死病毒中之VP3基因是如何誘發細胞凋亡及可能之分子機制。首先利用西方點墨法，發現在3T3細胞株內額外大量表現VP3至24小時中，發現可以誘導Bad基因表現，但對Bcl-2和Bax等基因則沒有明顯的影響。接著針對caspase-8和-3之受質來測試caspase-8和-3的活化情形。結果caspase-8在12小時明顯的大量被活化，同時caspase-3也在第24小時逐漸被活化，於72小時達到最高峰。接著利用專一性之caspase的抑制劑來抑制caspase-8及-3之活性，結果發現caspase-8及-3之專一性抑制劑在12和24小時有良好之抑制效果，並能阻段具有活性caspase-8與-3之形成和延遲細胞凋亡的現象產生。綜合以上的結果顯示，大量表現VP3能誘發3T3細胞株內的Bad蛋白質大量表現，及活化細胞內的caspase-8和下游的caspase-3，進而使得細胞走向凋亡。我們希望藉由此研究能進一步瞭解傳染性胰臟壞死病毒誘發細胞凋亡之分子機制，能有助於對傳染性胰臟壞死病毒所造成之疾病的預防和治療。 Abstract From the previous study, the Infectious Pancreatic Necrosis Virus IPNV could induces Chinook salmon embryo cell CHSE-214 death through apoptosis, but whether viral gene could involved in host cell death still don’t known. On the other hand, from our preliminary data shown that we found overexpression the vp3 could rapidly induce 3T3 cell death after 24hr incubation. So in this study, we want to examine vp3 how to induce apoptotic death and its molecular-death mechanism in 3T3 cells. Interestingly, we found that the bad gene could be induced at 24hr post-incubation time, but did not in Bcl-2 or Bax genes during vp3 overexpression in 3T3 cells. Then, initiator of caspase-8 and effector of caspase-3 also could be activated at either 12hr or 24hr post-incubation time, respectively. Finally, we found that specific caspase-8 and -3 inhibitors all could prevent the cell death and block the active forms of caspase-8 and -3 formation from precursor forms. In summary, the vp3 gene induces the 3T3 apoptotic cell death that may be mediated the pro-apoptotic gene bad expression to activate the caspase-8 and -3 for triggered the cell death, which could be useful for rational design of strategies for the prevention and /or treatment of IPNV induces diseases.