Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97732
標題: 開發創新性與高效電穿孔條件以使周邊血單核細胞重編程為誘導型萬能幹細胞
Developing a novel and efficient electroporation method for generating Human Induced Pluripotent Stem Cells from peripheral blood mononuclear cells
作者: 李奕可
Yi-Ko Lee
關鍵字: 周邊血單核細胞
誘導型萬能幹細胞
重編程
電穿孔
peripheral blood mononuclear cell
induced pluripotent stem cell
reprogramming
electroporation
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摘要: 胚胎幹細胞與誘導型萬能幹細胞(induced pluripotent stem cell, iPSC)具有自我更新與分化為各種細胞型態的能力,因此被認為是再生醫學研究的最佳實驗材料之一。2014年山中伸彌研究團隊使用無滋養層培養誘導型萬能幹細胞的系統而且顯示,以電穿孔方式重編程周邊血單核細胞中的T細胞,其iPSC產生效率為0.001~0.003 %。本實驗目的希望自行開發電穿孔的條件與緩衝液,兼具方便操作、成本低和無滋養層條件,來重編程周邊血單核細胞為誘導型萬能幹細胞,以應未來可以應用於臨床上的細胞治療。我們發現使用EM8代號的自製電穿孔緩衝液將3微克pCXLE –EGFP質體以電穿孔的方式送到新鮮周邊血單核細胞,可以達到約35-45 %的細胞存活率和40-60 %的螢光基因表現率。以上述方式將帶有Yamanaka 因子的質體(plasmid)以電穿孔方式送到周邊血單核細胞後,使用PHA/IL2活化增殖系統和無滋養層培養系統培養細胞,在第12天時可以觀察到類似胚胎幹細胞的細胞集落,培養到第20天之後,可以進行繼代。經過細胞集落的計算,使用此系統的iPSC產生效率可以達到0.001~0.015 %。產製的iPSC經過10代的繼代培養後,其型態表現正常,並且表現專一性標的蛋白,也具有擬胚體(embryoid body,EB)形成的能力和神經分化能力,並且透過設計專一性引子,使用PCR的方式確定在第五代後細胞無質體的殘留。我們希望以此系統產製誘導型萬能幹細胞,以應用未來幹細胞的相關研究。
Embryonic stem cells(ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent and can differentiate into every cell type.It serves as one of the best cell resources for regenerative medicine. In 2014, Yamanaka's research team developed a new system for the iPSC establishments, showing that T cells were reprogramed by electroporation, and the iPSC formation was 0.001 ~ 0.003 %. The purpose of my experiments is developing a novel and efficient electroporation condition for the T cell reprogramming into iPSCs.In our experiments, fresh peripheral blood mononuclear cells(PBMCs) were electroporated using homemade EM8 buffer. After the transfecting 3 µg of pCXLE-EGFP plasmid, the estimated cell viability was approximately 35- 45 % and 40- 60 % surviving cells exhibited the fluorescent gene expression.The PBMCs proliferation were primed by the PHA/IL-2 treatment.Serum-free ECO-M culture medium was added on the next day and ESC-like colonies began to emerge at the 12th day. Using this electroporation condition for the delivery of episomal transcription factors , we achieved a reprogramming efficiency up to 0.015 %.The iPSCs still expressed pluripotency markers and maintained a normal karyotype after 10 passages. They also had the ability to form embryonic body (EB) and differentiated into neural cells. At last, we used PCR to confirm the absence of plasmid integration in the cellular chromosome at the fifth generation.Taken together, these results demonstrated the feasibility of our electroporation system for the establishment of the iPSC.
URI: http://hdl.handle.net/11455/97732
文章公開時間: 2022-01-23
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