臺灣博碩士論文加值系統

利用轉染載體將誘導型多潛能性幹細胞(iPS細胞)轉錄因子(Oct4、Sox2、Klf4、c-myc)送入體細胞後，體細胞會因基因重組成而轉變成另一種細胞，此種細胞特徵與胚胎幹細胞類似，具有相同的基因表現與生長特性，同樣擁有自我更新且分化成三胚層細胞的能力，因此被命名為iPS細胞。目前最常用來製備iPS細胞的基因轉染方式為慢病毒載體，轉染效率僅約0.1%~1%。超音波是另一種新的轉染方式，當超音波在液體中傳遞時，會因空穴現象(cavitation)而產生真空小泡，真空小泡破裂時所釋放出來的壓力，能有效的攻擊細胞膜，增加細胞通透性，藉此增進基因轉染效率。
本研究先以不同粒徑磷脂體模擬細胞與反轉錄病毒，證實在超音波刺激下，能夠促進磷脂體通透性，並釋放內容物，以界面電位分析證實超音波並不會造成磷脂體化學組成的改變，利用calcein-磷脂體與GFP-慢病毒載體配合超音波作用於小鼠纖維母細胞，發現給予超音波刺激的組別，能促進calcein-磷脂體貼附在小鼠纖維母細胞上，並且GFP-慢病毒載體基因轉染率為23.81%，比未施加超音波的組別轉染率多了2.27倍，以iPSC–慢病毒載體感染小鼠纖維母細胞，發現有施加給予超音波的對照組比起未施加超音波的控制組，其iPS細胞聚落(colonies)數量增加了40%，觀察iPS細胞型態，並配合DNA電泳，免疫螢光染色法，拉曼圖譜分析，證實iPS細胞表現特有的細胞特徵，實驗結果發現超音波確實能增加慢病毒載體基因轉染率，進而增加iPS細胞聚落數，在未來可以利用超音波配合其他基因轉染方式，增強iPS細胞表現。

The somatic cell could reprogram to induced pluripotent stem cell (iPSC) by gene transfection vector. The characters of iPS cells are like embryonic stem cells (ESC), they have the same gene expression and life characteristic, include self-renew and differentiate to three germ layers. The preparation of iPSC is by lentiviral vector, but the transfect rate of is only 0.1 to 1%. Ultrasound is a new method of gene transfection. When ultrasound transmits energy in fluid, it could cause cavitations and damage the cell membrane. Therefore, ultrasound could enhance the cell permeability and then increase the gene transfecrion rate.
In this study, different sizes of liposomes were used to mimic cells and lentivirus. The permeability of liposomes was increased during insonation. Same phenomena were found for applying sound waves on mouse fibroblast in presence of calcein-liposomes and GFP-lentiviral vectors. Sound waves could enhance the binding of calcein-liposomes and increase the transfection of GFP-lentiviral vectors to fibroblast. The transfection efficiency of GFP-lentivires is 23.81% which 2.27-folds higher than that without ultrasound. Same protocol was employed for the transfection of iPSC. The iPS cellular colonies number is 40% higher than without sound treatment. The expression of iPS cells is then identified by cellular morphology, DNA electrophoresis, immunofluroescence and Raman microscopy. The result showed that ultrasound could enhance lentiviral vector gene transfection and then increase iPS cells colonies.

目錄
第一章 文獻回顧..................................................................1
1.1胚胎幹細胞簡介...........................................................1
1.1.1 胚胎幹細胞的特徵與歷史................................................1
1.1.2 胚胎幹細胞的應用與爭議................................................2
1.2多潛能性誘導型幹細胞的簡介.................................................4
1.2.1 多潛能性誘導型幹細胞細胞的歷史........................................4
1.2.2 多潛能性誘導型幹細胞在醫學上的發展.....................................5
1.2.3 多潛能性誘導型幹細胞在醫學上的應用.....................................6
1.3 多潛能性誘導型幹細胞轉錄因子介紹...........................................6
1.3.1 Oct4 (Octamer-binding transcription factor 4)簡介.................6
1.3.2 Sox2(SRY (sex determining region Y)-box 2)簡介....................7
1.3.3 Klf4(Kruppel like factor 4)簡介...................................7
1.3.4 c-Myc簡介.........................................................8
1.4 超音波(ultrasound)的介紹................................................9
1.4.1 超音波的歷史回顧....................................................9
1.4.2 超音波於生醫上的應用.................................................9
1.4.3 超音波參數在生醫上的應用.............................................10
1.5 拉曼光譜儀在細胞上的應用.................................................10

第二章 材料與方法...............................................................12
2.1 實驗材料..............................................................12
2.1.1 細胞株............................................................12
2.1.2 磷脂體製備........................................................12
2.2 實驗方法..............................................................12
2.2.1 以超音波刺激磷脂體..................................................12
2.2.1.1 超音波參數......................................................12
2.2.1.2 磷脂體率測定.....................................................13
2.2.1.3 磷脂體粒徑測量...................................................13
2.2.1.4 磷脂體界面電位測量................................................13
2.2.1.5 3T3細胞與calcein-磷脂體交互作用...................................14
2.2.2 細胞培養液製備.....................................................14
2.2.2.1 3T3細胞培養液...................................................14
2.2.2.2 STO細胞培養液...................................................14
2.2.2.3 餵養細胞前置培養液................................................15
2.2.2.4 iPS細胞培養液...................................................15
2.2.3 3T3細胞重組為iPS細胞與培養..........................................15
2.2.3.1 Cell counting kit-8 assay......................................15
2.2.3.2 餵養細胞製備.....................................................16
2.2.3.3 慢病毒載體轉染...................................................16
2.2.3.4 慢病毒載體轉染率計算..............................................16
2.2.4 iPS細胞特徵分析....................................................17
2.2.4.1 iPS細胞外型觀察與計算.............................................17
2.2.4.2 SEM觀察........................................................17
2.2.4.3 iPS細胞基因表現..................................................17
2.2.4.3.1 iPS細胞mRNA抽取...............................................17
2.2.4.3.2 Reverse transcription polymerase chain reaction (RT-PCR).....18
2.2.4.3.3 Polymerase chain reaction reaction (PCR) ....................18
2.2.4.3.4 DNA電泳法.....................................................18
2.2.4.3.5 免疫螢光染色...................................................19
2.2.4.3.6 拉曼圖譜分析...................................................20
2.2.4.3.7 Raman map....................................................20

第三章 實驗結果.................................................................21
3.1 超音波對磷脂體的影響....................................................21
3.1.1 超音波強度對磷脂體結構影響...........................................21
3.1.2 超音波頻率對磷脂體結構影響...........................................23
3.2 超音波對3T3細胞重組為iPS細胞的影響........................................24
3.2.1 超音波對3T3細胞的影響...............................................24
3.2.1.1 超音波對液體溫度的影響.............................................24
3.2.1.2 超音波強度對3T3細胞的影響..........................................24
3.2.1.3 超音波、3T3細胞與calcein-磷脂體間的交互作用..........................25
3.2.1.3.1 超音波強度、3T3細胞與calcein-磷脂體間的交互作用....................25
3.2.1.3.2 3T3細胞處理時間、超音波與calcein-磷脂體間的交互作用.................25
3.2.2 超音波與GFP-慢病毒載體間的交互作用....................................26
3.2.2.1 超音波強度對GFP-慢病毒載體轉染率的影響...............................26
3.2.2.2 3T3細胞處理時間對GFP-慢病毒載體轉染率的影響..........................27
3.2.3 3T3細胞重組為iPS細胞...............................................27
3.3 iPS細胞特徵分析........................................................28
3.3.1 iPS細胞型態觀察....................................................28
3.3.2 iPS細胞基因表現....................................................28
3.3.2 iPS細胞蛋白表現....................................................29
3.3.3 拉曼螢光譜儀分析....................................................29

第四章 討論....................................................................59
4.1 超音波與磷脂體的交互作用.................................................59
4.1.1 超音波強度與磷脂體..................................................59
4.1.2 超音波頻率與磷脂體..................................................60
4.2 超音波對小鼠纖維母細胞的影響..............................................60
4.2.1 超音波強度對小鼠纖維母細胞的影響.......................................60
4.2.2 超音波對小鼠纖維母細胞處理時間對基因轉染率的影響..........................61
4.2.3 超音波促進iPS細胞形成...............................................62
4.3 iPS細胞特徵分析........................................................62
4.4 結論及未來研究方向......................................................63

第五章 文獻回顧.................................................................64

表目錄
表2-1 primer序列...............................................................87
表2-2 PCR反應程序..............................................................88
表3-1 超音波強度對液體溫度增加的影響................................................58

圖目錄
圖2-1 超音波基因暨藥物傳送儀......................................................79
圖2-2 磷脂體製備與超音波刺激磷脂體釋放示意圖.........................................80
圖2-3 以1.05Mz超音波刺激磷脂體...................................................81
圖2-4 測量初始釋放速率示意圖......................................................82
圖2-5 iPS細胞反轉錄病毒載體......................................................83
圖3-1 磷脂體平均粒徑.............................................................31
圖3-2 超音波強度影響磷脂體初始釋放速率..............................................32
圖3-3 超音波強度影響磷脂體粒徑變化.................................................33
圖3-4 超音波強度影響磷脂體界面電位變化..............................................34
圖3-5 超音波頻率影響磷脂體初始釋放速率..............................................35
圖3-6 超音波頻率影響磷脂體粒徑變化.................................................36
圖3-7 超音波強度影響磷脂體界面電位變化..............................................37
圖3-8 超音波對3T3細胞的影響......................................................38
圖3-9 超音波強度影響3T3細胞吸附calcein............................................39
圖3-10 超音波強度影響3T3細胞吸附calcein螢光強度.....................................40
圖3-11 超音波處理3T3細胞時間影響calcein吸附........................................41
圖3-12 超音波處理3T3細胞時間影響calcein吸附螢光強度.................................42
圖3-13 超音波強度影響3T3細胞表現GFP...............................................43
圖3-14 超音波強度影響GFP-慢病毒載體轉染率...........................................44
圖3-15 超音波處理3T3細胞時間影響3T3細胞表現GFP......................................45
圖3-16 超音波處理3T3細胞時間影響GFP慢病毒載體轉染率..................................46
圖3-17 3T3細胞重組成iPS細胞......................................................47
圖3-18 超音波影響iPS細胞聚落(colony)數量..........................................48
圖3-19 3T3、STO與 iPS細胞外觀(morphology)型態....................................49
圖3-20 利用掃描式電子顯微鏡觀察iPS細胞.............................................50
圖3-21 Oct4與Sox2基因表現.......................................................51
圖3-22 Klf4與c-Myc基因表現......................................................52
圖3-23 利用免疫螢光法標記單一iPS細胞Oct4與Sox2蛋白..................................53
圖3-24 利用免疫螢光法標記iPS細胞群落Oct4與Sox2蛋白..................................54
圖3-25 拉曼顯微鏡觀察細胞型態.....................................................55
圖3-26 以拉曼圖譜分析3T3細胞與iPS細胞.............................................56
圖3-27 Raman map at 1374 (cm-1)...............................................57
圖4-1 以拉曼螢光光譜儀分析多潛能性幹細胞............................................84

附錄
表1-1 目前利用胚胎細胞研究的疾病...................................................82
表1-2 目前體細胞重組成iPS細胞的種類................................................83
圖1-1 體細胞重組成iPS細胞........................................................72
圖1-2 iPS細胞的應用.............................................................73
圖1-3 與Oct4互相影響的基因.......................................................74
圖1-4 Sox2與Oct4影響FGF4表現....................................................75
圖1-5 Klf4與其他基因的交互作用....................................................76
圖1-6 c-Myc基因促進胚胎幹細胞相關基因表現...........................................77
圖1-7 超音波促進基因轉染.........................................................78

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