利用缺氧狀態生成無病毒感染及致癌基因的老鼠誘導性多功能幹細胞

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Title:	利用缺氧狀態生成無病毒感染及致癌基因的老鼠誘導性多功能幹細胞 Mouse-induced pluripotent stem cells generated under hypoxic conditions in the absence of viral infection and oncogenic factors
Authors:	許千祐
Contributors:	基礎醫學研究所碩士班
Keywords:	誘導性多功能幹細胞;缺氧環境 iPS;hypoxia
Date:	2011-07-22
Issue Date:	2011-10-17 16:53:32 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	2006年，日本的一個團隊利用反轉錄病毒將Oct4, Sox2, c-Myc, Klf4這四個轉錄因子送入成體細胞，製作出誘導性多功能幹細胞。誘導性多功能幹細胞無論在各方法都和胚胎幹細胞十分相似，因此可以用來克服胚胎幹細胞在做臨床治療上所遇到困難。即使如此，誘導性多功能幹細胞仍有至少兩個需要克服的困難：1.使用病毒載體將基因插入成體細胞的染色體中 2.使用了致癌基因c-Myc, Klf4。在2008年，Okita等人報導他們可以在不使用病毒載體的情況下成功的生成老鼠的誘導性多功能幹細胞，但是他們在這個實驗仍然包含兩個致癌基因：c-Myc, Klf4。本實驗中我們的假說為：只使用兩個轉錄因子：Oct4, Sox2結合缺氧環境，應可以成功的生成誘導性多功能幹細胞。我們利用質體轉染重複將兩個表現質體（分別包含了Oct4, Sox2基因）傳入老鼠胚胎纖維母細胞當中生成誘導性多功能幹細胞，而沒有使用病毒載體。轉染六小時之後，讓細胞處於3%缺氧環境下二十四小時。這個動作總共重複了四次。在第九天將老鼠胚胎纖維母細胞培養到滋養層細胞上，在第二十一天時我們發現了誘導性多功能幹細胞。根據細胞的形態、鹼性磷酸酶染色、胚胎球實驗和畸胎瘤形成等實驗，我們證實了此誘導性多功能幹細胞具有多能性，和胚胎幹細胞相類似。此一方法不僅可以去除病毒感染的疑慮，也未使用致癌基因，因此對未來應用於臨床上有相當大的幫助。 Induced pluripotent stem (iPS) cells from somatic cells by introducting four transcriptional factors: Oct4, Sox2, c-Myc and Klf4 were reported in 2006. The morphology, proliferation, surface antigens, gene expression, telomerase activity, and epigenetic status of pluripotent cell-specific genes from the iPS cells were similar to those from embryonic stem (ES) cells. This novel technology has potential to overcome hurdles associated with ES cells due to their generation from mature somatic cells. However, at least two hurdles remain: integrating viral transgenes into the somatic genome, and introducing oncogenes: c-Myc and Klf4. In 2008, Okita et al. successfully generated mouse iPS cells without viral vectors, but they used four factors containing the oncogenes, c-Myc and Klf4. We tested our hypothesis that iPS cells can be generated by introducing only Oct4 and Sox2 under hypoxic conditions. We observed that the repeated transfection of two expression plasmids (one containing Oct4 and the other Sox2 cDNA) into mouse embryonic fibroblasts and combined hypoxic condition resulted in iPS cells generation. Six hours post-transfection, MEF cells were subjected to hypoxic conditions for 24 h (3% O2); this procedure was repeated four times. The MEF cells were passaged on feeder cells on day 9; iPS cell clones were observed 12 days post-passaging and designated as iPS-OSH cells. The morphology, positive alkaline phosphatase staining, embryonic body and teratoma formation indicated that the iPS-OSH cells had pluripotent capability similar to ES cells. This strategy avoids oncogenic factors and virus integration, thus decreasing the risk of cancer formation.
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