利用穿膜胜肽改善帶正電高分子之轉染效率

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林則緯(Tse-wei Lin) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

利用穿膜胜肽改善帶正電高分子之轉染效率
(The Use of Cell Penetrating Peptides and for Improving the Transfection Efficiency of Polyethylenimine)

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摘要(中)

本研究利用PEI(750 kDa & 25 kDa)結合穿膜胜肽作為基因載體，並以正負電荷吸引之方式，將之與質體DNA以不同胺基/磷酸根莫耳比(N/P ratio)組裝成奈米粒子。首先，由BCA assay測定細胞總蛋白量，我們發現R9與SAP10具有良好的生物適合性，然而IL在高濃度時具有明顯的細胞毒性。將PEI結合穿膜胜肽(Cell-penetrating peptides, CPP)作為基因載體能有效改善純PEI的轉染效率，以PEI/R9效率最佳。其中我們還發現在CPPs輔助下可更有效提升高分子量PEI(750 kDa)的轉染效率。我們將幾組轉染較佳的PEI與CPP結合的載體進行物理性質量測，由動態雷射散射粒徑分析儀與掃描式電子顯微鏡測得粒子之粒徑大小分佈於200 nm-400 nm之間；而表面電位介於10~20 mV，均為適合基因傳送之條件。以電泳方式簡單測試其包覆率，可發現粒子表層的PEI能將質體DNA完整包覆形成穩定的粒子。藉由改變合成粒子的順序，我們證實轉染的增進主要是基於複合於粒子上的CPPs的效果。最後，我們探討CPP輔佐轉染的機制，發現此系統CPPs不但可以促進複合體進入細胞的效率(尤其是R9)；還能夠輔助複合體以質子海綿效應以外的路徑脫離內胞(endosome)；另外藉由競爭實驗，我們證實IL及SAP10可以增加質體DNA與載體分離的機會。因此，透過本研究我們了解CPP可以幫助陽離子高分子進行基因傳送，這些結果將有助於發展安全且高效率的非病毒載體以應用於基因治療。
關鍵字：聚乙烯亞胺、穿膜胜肽、基因傳送

摘要(英)

In this study, we would like to investigate if the combination cell penetrating peptides (CPPs) and polyethylenimine (PEI) may facilitate gene delivery. Polyethylenimine (PEI, 750 kDa & 25 kDa) were combined with cell-penet rating peptides(CPPs) as gene vectors. By electrostatic interaction, self- assemble nanoparticles using PEI, CPPs, and plasmid DNA were prepared in different amine/phosphate (N/P) ratios. The BCA assay was applied to evaluate the cytotoxicity of CPPs. Both R9 and SAP-10 were biocompatible compared to IL which may induced serious toxicity at high concentration. By combing PEI and CPPs as gene vehicles, transfection efficiencies can be highly improved over the pure PEI delivery, which was especially efficient to promote gene transfer of PEI with molecular weight of 750 kDa, a poor vector when it was solely administrated. Dynamics light scattering (DLS) assay and scanning electron microscopy (SEM) were performed to characterize PEI/CPPs/DNA complex. Complexed particle demonstrated sizes between the 200 nm to 400 nm and zeta potentials between +10 to +20 mV, suggesting that these nanoparticles should be suitable for gene delivery. The electrophoresis data indicated that the PEI and CPPs could complex DNA completely to form a stable particle. The experiment of changing complexation order revealed that only complexed CPPs may help DNA delivery. Finally, the delivery mechanism of CPPs was studied. It suggested that CPPs should not only increase cellular uptake, but also promote complexes escaping from endosome by route other than proton sponge effect. In addition, the competition experiment demonstrated that IL and SAP10 should interfere the interaction between PEI and DNA to increase the chance of dissociation when they were internalized. Through this study, the adjuvant effects of CPPs on facilitating gene delivery using cationic polymer were studied, which should be beneficial to development a safe and highly efficiency nonviral vector for gene therapy.
Key words：polyethylenimine, cell-penetrating peptides, gene delivery

關鍵字(中)

★ 基因傳送
★ 穿膜胜肽
★ 聚乙烯亞胺

關鍵字(英)

★ gene transfer
★ cell penetrating peptides
★ polyethylenimine

論文目次

目錄
摘要 I
Abstract III
目錄 V
圖目錄 IX
第壹章序論 1
1-1 背景 1
1-2 實驗動機 2
1-3 實驗目的 5
1-4 實驗假設 5
第貳章文獻回顧 8
2-1基因傳送療法 8
2-2基因載體 12
2-2-1脂質體 13
2-2-2正電高分子 14
2-2-3以胜肽進行基因輸送 17
2-2-4結合PEI與胜肽 21
2-3本研究方向 22
第參章實驗材料與方法 23
3-1試藥與原料 23
3-1-1質體DNA 23
3-1-2細胞 25
3-1-3穿膜胜肽(Cell-penetrating peptides) 25
3-1-4藥品 26
3-2儀器 27
3-3試藥配製 28
3-4質體DNA純化 29
3-5 HEK293T和NIH-3T3細胞培養 32
3-6 PEI結合不同種類CPP對轉染效率影響 35
3-6-1細胞轉染 35
3-6-2 ONPG分析 36
3-7 轉染機制探討 39
3-7-1細胞吸收(uptake)效率(以Fluorescein標記DNA) 39
3-7-2外加CPPs的效應 41
3-7-3 Bafilomycin A1作用 42
3-7-4 外加alginate競爭效應 42
3-8奈米粒子製備及物理化學性質鑑定 44
3-8-1 奈米粒子製備 44
3-8-2雷射粒徑分佈儀(dynamic light scattering, DLS)分析 44
3-8-3 掃描式電子顯微鏡(scanning electron microscope, SEM) 45
3-8-4包覆率分析(膠體電泳分析法) 46
3-9 實驗架構 47
第肆章結果與討論 48
4-1 PEI結合穿膜胜肽對細胞毒性影響 48
4-2 PEI結合穿膜胜肽對轉染效率影響 54
4-3 奈米粒子物理性質鑑定 61
4-3-1粒徑大小(DLS測量和SEM觀測) 61
4-3-2表面電位(DLS測定) 65
4-3-2包覆率(電泳) 68
4-4 CPPs形式對於轉染的影響 70
4-4-2外加CPPs實驗 72
4-5 轉染機制探討 74
4-5-1細胞吸收效率 74
4-5-2內胞脫離作用(endosomal escape)之抑制劑影響 77
4-5-3 載體與質體DNA 解離(dissociation)行為 80
第伍章結論 83
第陸章參考文獻 85
圖目錄
圖1-1 PEI(25 kDa)轉染HEK293T細胞結果 3
圖 1-2 PEI與其合成物對於HEK293T細胞的毒性測試 3
圖1-3 實驗假設1 5
圖 1-4 實驗假設2 6
圖1-5 實驗假設3 7
圖 3 - 1 pCMV-β結構圖 23
圖 3 - 2 pEGFP-C3結構圖 24
圖4-1 BCA assay原理 49
圖4-2 BCA assay：CPPs-DNA結合PEI(750 kDa)後對NIH-3T3細胞之存活率影響(a)R9 (b)IL (c)SAP10 52
圖4-3 BCA assay：CPPs-DNA結合PEI(25 kDa)後對NIH-3T3細胞之存活率影響(a)R9 (b)IL (c)SAP10 53
圖4-4 β-galactosidase assay反應圖 54
圖4-5 beta-galactosidase assay：純CPPs與CPPs-DNA結合PEI(750 kDa)後對NIH-3T3細胞之轉染效率(a) pure CPPs (b) PEI/R9 (c)PEI/ IL (d)PEI/SAP10 57
圖4-6 beta-galactosidase assay：CPPs-DNA結合PEI(25 kDa)後對NIH-3T3細胞之轉染效率(a)PEI/R9 (b)PEI/IL (c)PEI/SAP10 58
圖4-7 比較兩種不同分子量PEI結合CPPs作載體，分別轉染HEK-293T與NIH-3T3細胞之轉染效率 60
圖4-8 各種類CPPs結合DNA與PEI後粒子之粒徑變化 63
圖4-9 SEM觀測結果 63
圖4-10 CPPs-PEI(750 kDa & 25 kDa)-DNA粒子之表面電位(a)R9 (b)IL (c)SAP10 67
圖4-11 不同CPPs與DNA結合之粒子和結合PEI(750 kDa & 25 kDa)後粒子之電泳圖(a)R9 (b)IL (c)SAP10 69
圖4-12 不同順序合成的粒子對於NIH-3T3細胞的轉染結果比較 71
圖4-13 不同合成粒子順序對粒子表面電位影響 71
圖4-14 外加自由的穿膜胜肽對於轉染結果影響(a)R9 (b)IL (c)SAP10 73
圖4-15 細胞吸收效率結果(40X) 76
圖4-16 Bafilomycin A1對轉染效率的影響 79
圖4-17 測試結合CPPs之載體和純PEI與DNA的結合強度(alginate競爭效應) 82

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指導教授

胡威文(Wei-wen Hu)

審核日期

2012-8-23

推文