建立以重組蛋白生產安全之自體多能性幹細胞的方法;Formulating a protocol for generating safe pluripotent stem cells from somatic cells by recombinant proteins

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题名:	建立以重組蛋白生產安全之自體多能性幹細胞的方法;Formulating a protocol for generating safe pluripotent stem cells from somatic cells by recombinant proteins
作者:	趙冠豪;Zhao,Guan-Hao
贡献者:	生命科學系
关键词:	誘導性多功能幹細胞;蛋白轉導區塊;重新編成;induced pluripotent stem cells;protein transduction domain;Reprogramming
日期:	2014-07-29
上传时间:	2014-10-15 14:38:55 (UTC+8)
出版者:	國立中央大學
摘要:	利用特定轉錄因子將體細胞轉變為誘導多能性幹細胞在再生醫學上有令人期待的前景。至今已有許多方法能產生誘導多能性幹細胞，例如使用反轉錄病毒、慢病毒、質體、轉位子、合成信使核醣核酸以及重組蛋白。然而，外送的基因物質可能造成不預期的基因組突變。相較之下，以送入重組蛋白產生多能性幹細胞的方式可以避免去氧核醣核酸隨機插入的可能性。在這篇研究中，我們利用聚精胺酸蛋白轉導區塊來送達重組的重新編成因子。五個重新編成因子：Oct4,Sox2,Nanog,Lin28和c-Myc的羧基端都黏合上聚精胺酸蛋白轉導區塊。我們能將這些因子順利的表達並純化；然而，Oct4和Nanog蛋白的表現量相對來說非常低。令我們驚訝的是，將MyoD轉活區塊黏合在Oct4和Nanog的氨基端顯著性的增加蛋白表現。這些蛋白表現在大腸桿菌的包涵體或是原生型態都可以被溶解、重新摺疊並以鎳螯合物凝膠純化。純化的蛋白能轉導通過細胞膜並轉移至細胞核。現今我們正以這些蛋白組合一些小分子化合物，如丙戊酸，來測定最適合重新編成體細胞轉變為誘導多能性幹細胞以及種系特異性幹細胞的條件。;Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by defined factors has showed hopeful perspective in regenerative medicine. Currently, many approaches are employed to generate iPSCs, including retroviruses, lentiviruses, adenoviruses, plasmids, transposons, synthetic mRNAs and recombinant proteins; however, the introduction of ectopic genetic materials may cause unexpected genetic modifications. In contrast, delivery of recombinant proteins can avoid the possibility of random DNA integration. In this study, we utilized poly-arginine (poly-R) protein transduction domain (PTD) for delivery of recombinant reprogramming factors. The PTD was fused to the C-terminus of five reprogramming factors: Oct4, Sox2, Nanog, Lin28 and c-Myc. These chimera factors could be expressed and purified with normal function in E.coli; however, the expression of Oct4 and Nanog was relatively low. To our surprise, fusion of the powerful MyoD transactivation domain to the N-terminus of Oct4 and Nanog significantly enhanced the protein yield. These proteins expressed in E.coli as inclusion bodies or native forms were solubilized, refolded, and purified using Nickel-Chelating Sepharose. These purified proteins were able to transduce the plasma membrane and translocate into nucleus. Currently, we are combining these proteins and small compounds, such as valproic acid, to determine the optimal condition for reprogram somatic cells into iPSC and lineage-specific stem cells.
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