臺灣博碩士論文加值系統

　　細胞核重整(reprogramming)乃為改變已分化細胞核的基因表現，使之表現不同譜系的細胞特徵，或恢復多能性甚至於全能性的分化潛力。過去認為已分化的細胞核，僅能表現特定功能。但近來由細胞核轉置的研究證實可將以分化體細胞核去分化，並且能如胚幹細胞或受精卵般發展出完整個體。此種藉由卵細胞質去分化的能力，在異種體細胞核/卵母細胞質也能發生，例如：豬核/牛卵、羊核/兔卵。說明了這樣去分化的現象及參與的分子機制在不同物種間亦極為相近，也表示細胞核表現會受到周圍細胞質環境所影響，進而改變原來細胞特性。此外，藉由改變細胞膜通透性將培養液中所加入的胸腺淋巴細胞萃取液(T-lymphocyte lysate)導入小鼠成纖維細胞(fibroblast)，此時成纖維細胞會由原來呈紡錘狀的的細胞型態會變得與胸腺淋巴細胞型態極為相似，且成纖維細胞的分子細胞標誌也發生消退及改變，而逐漸呈現胸腺淋巴細胞的細胞標誌。
　　在本實驗中，我們利用上述膜通透性改變的方式，將小鼠胚幹細胞的細胞萃取液導入小鼠及人類成體成纖維母細胞，結果可發現處理後的細胞型態隨著培養的時間，紡錘狀的外型會消失而產生類似胚幹細胞的團簇狀(colony formation)。由免疫染色結果證明原本屬於中胚層已分化的成纖維細胞，經過處理後可表現屬於未分化胚幹細胞的分子標記，如： Alkaline phosphatase (AP)活性，並且具有轉分化(trans-differentiation)成其他胚層細胞譜系的能力。利用即時定量聚合連鎖反應也可發現經導入小鼠胚幹細胞萃取液的小鼠及人類的成纖維細胞能表現只能在分化狀態的胚幹細胞才能表現的Oct-4、Nanog及Pecam-1等相關基因。從本論文的試驗結果得知：導入以胚幹細胞萃取液到體細胞中的方式，的確可以使得分化末期的體細胞進行細胞核重整，且產生型態與基因表現皆近似於胚幹細胞的去分化現象。期待在未來可用此便利之方式產製大量的幹細胞供日後研究或醫療使用。

　Nuclear reprogramming is defined as altering the gene activity of differentiated cells to express the characteristics and function of different lineage, even resume their pluripotency. Several researches have demonstrated that nuclear reprogramming is possible. Nuclear transfer with somatic cells had proven to be able to give rise to cloning animals and derivation of embryonic stem cells. Induction of plasticity of adult stem cells could promote somatic cells to trans-differentiate into other cell lineages.
　After increasing permeability of the plasma membrane and introduction of ES-D3 cell extract, the morphology of somatic cells changed into a ES cell-like shape. Quantitative real-time PCR analysis showed both reprogrammed Dunni cells and HS-68 cells expressed Oct-4, Nanog and Pecam-1 genes which are stage-specific to undifferentiated ES cells. Strong alkaline phosphatase (AP) activity was also observed in reprogrammed cells. Under spontaneous differentiation, reprogrammed somatic cells could trans-differentiate into different lineages of three primary germ layers. According to our results, terminal differentiated human and mouse fibroblasts can be reprogrammed by mouse embryonic stem cell extracts. However, the protocol should be further modified to achieve a better efficiency on reprogramming.
　In the present study, we found that the cellular characteristics of somatic fibroblast cells derived from mouse skin (Dunni cells) and human foreskin (HS-68) could be reprogrammed by the cell extract of mouse embryonic stem cells (ES-D3).

中文摘要 ……………………………………………………………I
Abstract …………………………………………………………… II
Acknowledgement…………………………………………………III
Abbrevations ……………………………………………………… IV
Table of Contents………………………………………………… VI
List of Illustrations………………………………………………VIII
List of Appendixes…………………………………………………IX
References Review ………………………………………………… 1
1. Cell reprogramming …………………………………………..…1
1.1 Progress of reprogramming …………………………….… 1
1.2 Advanced reprogramming－interspecies cloning…………….2
1.3 Techniques to approach reprogramming………………………3
1.3.1 nuclear transfer…………………………….…………… 3
1.3.2 cell-cell fusion ……………………………….………… 3
1.3.2.1 ESC-somatic cell fusion…………………………….4
1.3.2.2 EGC-somatic cell fusion ……………………………4
1.3.2.3 ECC-somatic cell fusion…………………………….5
2. Cell-extraction based reprogramming…………………….……….. 6
2.1 Introduction …………………………………………………..…6
2.2 Applications and benefits of cell-extraction
reprogram……………………………………….………………7
2.3 Streptolysin-O mediated reversible
permeabilization……………………………….………………8
Materials and Methods ………………………………………………. 9
1. Cells ………………………………………………… …………..9
2. Preparation of cell extracts...……………………………………..9
3. Permeabilization of Plasma Membrane of Somatic cells.……….10
4. Reprogramming the Somatic Cells with Stem Cell Extracts..…. 10
5. Quantitative Reverse Transcription-Polymerase Chain
Reaction ……….………………………………………………...10
6. Immuno-fluorescent staining procedures ……………………… 11
7. Alkaline phosphotase detect procedures ……………………… .11
Results …………………………………………………………………..12
1. Cell membrane reversible permeabilization ……………………12
2. Preparation of stem cells extracts ……………………… …….. 12
3. Morphological changes of reprogrammed cells ………………. 12
4. Quantitative Reverse Transcription-Polymerase
Chain Reaction ……………………… …………………………13
5. Alkaline phosphatase activity assay ……………………… …... 14
6. Immuno-histochemistry assay …………………………………. 14
Discussion ………………………………………………………………16
Conclusion ………………………………………………………………20
References ………………………………………………………………33

List of Illustrations
1. Figure 1. Streptolysin-O reversible permeabilized cells membrane...21
2. Figure 2. Preparation of stem cells extracts…………………………22
3. Figure 3. Morphological changes of rDunni
(reprogrammed Dunni) cells……………………………………….. 23
4. Figure 4. Morphological changes of rHS-68
(reprogrammed HS-68) cells ………………………………………. 24
5. Figure 5. Embryoid-like cell clusters appeared at Dpr=11………….25
6. Figure 6. β-actin as control curve…………. …………. …...……….26
7. Figure 7. Oct-4 gene expression in extract-treated somatic cells .…..27
8. Figure 8. Nanog gene expression in extract-treated somatic cells. ....28
9. Figure 9. Pecam-1 gene expression in extract-treated somatic cells. .29
10. Figure 10. Reprogrammed somatic cell express significant alkaline phosphatase…………. …………. ……………. …………………...30
11. Figure 11. Reprogrammed Dunni cell immuno-fluorescent stain … .31
12. Figure 12. Reprogrammed HS-68 cell
immuno-fluorescent stain ……………. …………. ………………. 32

Appendixes
1. Appendix 1. Streptolysin-O structure …………. …………. ………39
2. Appendix 2 List of clone animals..………. …………. ………….. 40

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