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標題: 利用昆蟲桿狀病毒表現載體研究哺乳動物細胞株之基因轉導
The Use of Baculovirus Expression Vector for Studying The Gene Transduction of Mammalian Cells
作者: Jessica, C.Y.Lu
出版社: 昆蟲學系所
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摘要: The present study used baculovirus expression vector system that is based on the budded form of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) to construct recombinant viruses, vAtEsL, carrying three promoters, i.e., CMVie-enhance promoter, p10 promoter, and T7 promoter, to drive the enhanced green fluorescent protein (EGFP) as a reporter and another recombinant virus carrying a survivin promoter to drive luciferase. There were different transduction rates among 19 mammalian cell lines. The bone cancer cell line (U2-osl) had the highest transduction rate followed by normal oral cell line (NHOK) and oral cancer cell lines (OECM-1, SAS and OC3). This finding could possibly be applied to study of oral cancers or to be used as its early diagnostic tool. The survivin promoter expressed successfully in the plasmid (pActEsB) to drive bax gene and triggered apoptosis; whereas its constructed recombinant baculovirus, vActEsB, was incapable of expressing it being due to low transduction rate, gene homology and/or variation in construction strategy. The other recombinant baculovirus, viz., vFuPiEG, was constructed based on FasBacI vector containing ubiquitin promoter to drive progranulin (PGRN) gene and internal ribosome entry site (IRES) to express EGFP. Since PGRN was expressed at a sufficient amount in H293 cells endogenously, its over-expression could not help to study the relationship between PGRN and NF-κB in inflammation. Therefore, this study underwent siRNA treatment to knock down PGRN expression in H293 cells, resulting in decrease of NF-κB activity. In addition, treatment of C6 malignant glioma cells and normal glial cells from rat with PGRN siRNA exhibited effect of PGRN on Akt activity. The results showed that PGRN knockdown was able to increase total Akt expression but to decrease phosphorylated Akt in the normal glial cells. Conversely, this knockdown did not obviously influence the glioma cells. Based upon the preliminary results in this study, it seems that the baculovirus expression vector system could potentially be employed in investigations on human cancer diseases and inflammation.
本文利用加州苜蓿夜蛾核多角體病毒(Autographa californica nuclopolyhedrovirus, AcMNPV )表現載體系統,構築含CMVie-enhance promoter、p10 promoter及T7 promoter等三個promoters驅動水母螢光基因(EGFP)及survivin promoter驅動Luciferase基因之重組桿狀病毒(vAtEsL),在感染19種之哺乳動物細胞株中,發現其在不同哺乳動物細胞之轉染力不一,以骨癌細胞(U2-osl)的轉染力最佳,口腔正常細胞(NHOK)與口腔癌細胞(OECM-1、SAS及OC3)次之,此對口腔癌可以應用於研究其致病機制或早期診斷之工具。另外,構築以survivin promoter驅動bax基因之重組桿狀病毒,結果其可藉由重組桿狀病毒的質體(pActEsB)在癌細胞株表現,且引起細胞凋亡,惟組成桿狀病毒(vActEsB)後,卻因轉染力不穩定或太低,以及在重組期間homology問題,質體構築設計之差異等因素,造成bax基因在轉染後並無理想的表達。本文的下半部份集中於探討醣基化蛋白 PGRN與哺乳動物內訊息傳遞之關係,雖先以FasBacI vector為基礎所構築的重組桿狀病毒(vFuPiEG),以ubiquitin promoter驅動醣基化蛋白 PGRN,並用IRES 啟動EGFP的報導基因,但由於試驗過程中發現H293細胞內的PGRN本底表達已經足夠,重組桿狀病毒所生產的PGRN無助於對該蛋白的功能性探討。因此,本試驗進行siRNA處理,對H293細胞之本底PGRN進行knockdown後,則發現NF-κB 的活性會隨PGRN之降低而下降。另外,以PGRN siRNA處理老鼠腦膠質瘤細胞(glioma cell,C6)與正常膠質細胞(glial cell),初探PGRN knock down後在Akt pathway之反應,試驗結果發現在正常的膠質細胞,經PGRN knockdown後Akt表達會上升,而磷酸化的AKT則降低;但在腫瘤細胞上, PGRN knockdown後對Akt通路無明顯影響。本文之初步結果顯示昆蟲桿狀病毒表現載體,在人類癌症及炎症之研究上似具有應用之潛力。
其他識別: U0005-1407200911203800
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