本研究主要探討桿狀病毒是否能有效的傳遞基因至老鼠關節軟骨細胞或人類間葉幹細胞(mesenchymal stem cells，MSC)，並應用於軟骨組織工程之基因治療。我們首先利用帶有綠色螢光蛋白EGFP和CMV-IE啟動子的重組桿狀病毒(Bac-CE)探討轉軟骨細胞之轉導效率和外來基因表現。若以傳統方式(37 °C、MOI=200、以培養液為轉導環境溶液作用1小時)進行轉導，轉導效率相當低(< 5 %)。因此我們改變轉導條件(25 °C、以PBS作為轉導環境溶液作用8小時)，轉導效率可提高至88 %並延長基因表現時間至21天。以此條件轉導(轉導時間為4小時)人類臍帶血(uMSC)和骨髓(bMSC)間葉幹細胞，效率可高達70 %以上。以即時定量聚合酶連鎖反應(Q-PCR)分析發現，在25 °C、以PBS作為轉導環境溶液、延長細胞和病毒作用的時間，能夠增加病毒進入細胞的基因含量，並有效的提升轉導效率、基因表現量及時間。轉導過之軟骨細胞和間葉幹細胞，其生長速率會因EGFP表現而減緩，但會隨病毒DNA含量和EGFP表現量遞減而逐漸回復，並且在轉導後軟骨細胞仍可正常分泌細胞外間質(醣胺素和第二型膠原蛋白)，而MSC則可繼續分化成脂肪細胞。此外，利用重複轉導(super-transduction)和細胞重新繼代再轉導(re-transduction)方式，也有效的提升轉導效率和延長EGFP表現時間。
更重要是，我們亦發現桿狀病毒可以轉導進入不同分化路徑(脂肪、軟骨和骨細胞)及分化階段(分化後第1、2、3和4週)的間葉組織的前驅細胞，在往脂肪細胞路徑分化後1週轉導有最高的轉導效率(90 %)和表現時間(41天)。其轉導效率和基因表現(時間)會隨前驅細胞分化路徑和分化階段有顯著的差異，依序為脂肪細胞>骨細胞>軟骨細胞，這些差異與培養基添加物(Dexamethasone或A酸)、細胞週期以及病毒進入細胞的基因數無關，而由egfp轉錄程度不同所致。以化學組織染色和反轉錄聚合酶連鎖反應(RT-PCR)分析，在多種不同分化階段之前驅細胞其分化能力仍不受病毒轉導影響。綜合上述，桿狀病毒可以有效傳遞基因至老鼠關節軟骨細胞和人類間葉幹細胞並做為軟骨組織工程之基因治療載體。

To assess the potential of baculovirus as a gene delivery vector for cartilage tissue engineering, a recombinant baculovirus expressing enhanced green fluorescent protein (EGFP) was constructed to transduce rat articular chondrocytes and human mesenchymal stem cells (MSCs). Using the traditional transduction protocol (incubating the cells with concentrated virus at a multiplicity of infection (MOI) 200 for 1 h), the gene delivery efficiency into chondrocyte was low (<5 %). Therefore, a modified protocol was adopted (incubating cells with unconcentrated virus for 8 h with PBS as surrounding solution), which markedly enhanced the efficiency (88 %) and prolonged the duration of expression (21 days). Using the transduction condition we developed, MSCs were transduced at efficiencies up to 70 %. The elevated efficiency, gene expression level and duration correlated well with the increased virus uptake upon extended incubation time at 25 C. Although the virus transduction somewhat hindered the cell proliferation due to EGFP overexpression and the accompanying metabolic burden, the growth rate could restore when the burden was relived in the long-term culture. More importantly, the virus transduction imposed no perceptible cytotoxicity and the chondrocytes could secrete articular cartilage-specific type II collagen and glycosaminoglycan as well as mock-transduction cells did. Additionally, baculovirus-transduced MSC remained capable of differentiating into adipocytes.
Furthermore, baculovirus could transduce MSC progenitor cells differentiating into different lineages (adipogenic, chondrogenic and osteogenic) at different differentiation stages (1, 2, 3 and 4 weeks post-induction). For MSC induced into adipogenic pathway, the transduction at 1 week post-induction resulted in an efficiency of up to 90 % and a duration of transgene expression for up to 41 days. Notably, the transduction efficiencies and transgene expression durations varied with the differentiation lineage and differentiation stages. The variation in transgene expression was independent of the cell cycle and virus entry, but resulted from the differential efgp transcription in the progenitors differentiating along different pathways. The differentiation capacities of MSC progenitors were not influenced by baculovirus transduction, as demonstrated by histochemistry staining and reverse transcription PCR (RT-PCR). These data collectively confirmed that baculovirus enables highly efficient gene transfer into rat articular chondrocytes and human MSCs, but does not impair the normal proliferation and differentiation capability of the transduced cells, suggesting that baculovirus expressing growth factors may be used to genetically modify, and modulate the differentiation status of chondrocytes and MSCs.

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