雖然目前已有許多研究報導以桿狀病毒作為載體可將基因送入哺乳動物細胞，然而研究多偏向以桿狀病毒作為基因治療載體的應用，對於最佳的轉導條件則未有系統性的研究。本研究中我們提出使用未濃縮的桿狀病毒將基因送入HeLa細胞株的轉導方法。在轉導時我們取出培養基後將細胞加入病毒液，以磷酸緩衝溶液(D-PBS)作為轉導環境溶液於室溫下進行轉導4 h，可表現報導基因並達到75-85 %的轉導效率。相較於其他研究團隊所提出將細胞與濃縮的病毒以培養基為環境溶液，於37 °C轉導1 h的方法，本研究提供的方法既簡單又有效率。經由以上最適化轉導條件研究我們發現：(1)將高劑量的病毒與HeLa細胞株進行轉導至少4 h可得到良好的轉導效率；(2)於37 °C下，桿狀病毒的半生期會明顯地降低；(3)當細胞生長到多層時，使用EGTA前處理無法明顯地增加感染效率；(4)相較於TNM-FH與D-PBS，DMEM不適合作為轉導時的環境溶液，因此我們推測DMEM中含有抑制轉導效率的因子。關於DMEM造成低轉導效率的原因，本研究中測試了數種可能的因素，如pH、陽離子效應及glucose濃度等，目前仍未發現DMEM中影響轉導效率的因素；(5)使用本研究中所提出的轉導方法雖然與一般細胞培養及利用病毒轉導的條件相差甚大，對細胞生長只有短暫的影響，對細胞的存活率沒有明顯的影響，也不會出現細胞毒性。本研究提出了省略濃縮步驟時，桿狀病毒/哺乳動物細胞表現系統的最佳轉導策略，經由此策略可成功地將重組基因送入哺乳動物細胞中，並得到與其他研究者相似的轉導結果。經由這樣的研究結果，成功的避免了超高速離心可能對病毒活性造成的影響及大量生產時超高速離心程序帶來的不便。

Although baculovirus-mediated gene delivery into mammalian cells has been reported in many literatures, previous studies were focused on the application of baculovirus as a gene therapy vector. Besides, systematic investigation of the optimal transduction conditions remains unavailable. In this work, a transduction protocol using unconcentrated baculovirus was proposed for simple and efficient gene delivery into HeLa cell. We found that □75-85% of the cells could be readily transduced and express the reporter protein when virus transduction occurred for 4 h at 25□C using Dulbecco’s phosphate-buffered saline (D-PBS) as the surrounding solution. This method contrasted to previous protocols in which transduction occurs for 1 h at 37□C using the growth medium (e.g. DMEM) as the surrounding solution. Investigation of the physical parameters led to the finding that (1) baculovirus uptake by HeLa cells continued for at least 4 h in the event of high virus dosage, which led to higher gene expression; (2) the half-life of baculovirus dramatically decreased at 37□C; (3) EGTA pretreatment did not apparently facilitate the gene delivery when the cells grew to multilayers; (4) lower transduction efficiency and gene expression were obtained when DMEM was used (in comparison with D-PBS and TNM-FH), suggesting that DMEM contains certain inhibitory factors for baculovirus transduction; several factors (e.g. pH, ions, and glucose concentration) were tested to investigate the low efficiency caused by DMEM, but the reasons were not clear yet; (5) the transduction conditions we proposed was totally different from the usual culture conditions and the transduction conditions used by other groups. However, this protocol did not cause cytopathic effect but only transient effect on grow curve. Our data uncovered several aspects that were not investigated before and the optimized transduction conditions allowed for gene delivery as efficient as that by the protocols commonly employed by others, but eliminated the need for virus ultracentrifugation. The protocol not only represented a simpler approach, but also considerably reduced possible virus inactivation during ultracentrifugation, thus making it easier to convert the baculovirus/mammalian cell system to a tool for eucaryotic protein production in a larger scale.

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