桿狀病毒是目前新興的基因治療載體，而誘導性多功能幹細胞近年來也被視為在再生醫學上的應用有極大的潛力，目前已有利用腺病毒以及添加生長因子或是其他方式，以胚胎幹細胞所建立的分化方式成功的使誘導幹細胞分化成許多種類的細胞，包括神經細胞、造血幹細胞等。目前尚未有文獻指出可以利用桿狀病毒攜帶外源基因轉導誘導性多功能幹細胞，而由於已有文獻證實桿狀病毒可以成功的轉導胚胎幹細胞，又由於胚胎幹細胞與誘導性多功能幹細胞在基因表現、表面標記蛋白、分化功能上諸多的相似性，於是本實驗室認為建立一個轉導誘導性多功能幹細胞的系統是可行的，並且期望利用桿狀病毒攜帶外源基因的方式應用於誘導性多功能幹細胞的分化。本研究可分為兩個主軸，第一部分建立桿狀病毒轉導誘導性多功能幹細胞之系統，在轉導策略的部分我們首先探討桿狀病毒攜帶不同啟動子對於外源基因表現的差異，研究結果發現在CAG、CMV、EF1-α三者啟動子中，CAG啟動子在添加丁酸鈉(sodium butyrate)後驅動外源基因的表現明顯高於其餘兩啟動子；後續進行最適化所添加的丁酸鈉濃度，比較添加不同濃度的丁酸鈉對於增加外源基因表現及後續形成類胚囊體的影響，發現在添加較高丁酸鈉濃度時發現會有影響類胚囊體形成效率的影響，於是在外源基因表效率及類胚胎體效率中取平衡點。進一步我們也研究利用轉導iPS細胞適當的時機點，以延長短效桿狀病毒所攜帶的外源基因之表現。第二部分我們設計五株病毒，同時表現生長因子BMP4、VEGF和SCF及轉錄因子AML1B和SCL，誘導iPS細胞分化成造血幹細胞。利用ELISA和RT-PCR分析外源基因表現量，並利用流式細胞儀分化效率。目前尚未有文獻指出利用桿狀病毒載體誘導iPS 細胞分化成造血幹細胞，故本研究具有其價值性。

Baculovirus is a promising vector for gene therapy while induced pluripotent stem (iPS) cells hold great potential in regenerative medicine. It is reported that baculovirus can successfully transduce embryonic stem cells, yet whether baculovirus can transduce iPS cell remains unknown. Due to the similarity of gene expression, surface markers and differentiation functionality between embryonic stem cells and iPS cells, creating a system to transduce iPS cells is feasible. This study aims to employ baculovirus to deliver appropriate transgenes into iPS cells in order to induce iPS cells differentiation into hematopoietic progenitors. We first constructed the recombinant baculoviruses carrying the transgenes and then evaluated the gene expression mediated by different promoters and the dosage effect of sodium butyrate. We found that in iPS cells CAG promoter drove the gene expression better than the CMV and EF1-α promoter, and discovered that high dosage of sodium butyrate affected embryonic body forming. Second, we constructed five different viruses carrying different genes encoding various growth factors and transcription factors. After virus transduction of iPS cells, we analyzed the transgene expression and the efficiency of hematopoiesis differentiation by ELISA, RT-PCR and flow cytometry. This study can open a new avenue to direct the iPS differentiation toward the hematopoietic lineage.

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