利用生物素改質之幾丁聚醣進行基因原位傳送｜國立中央大學博碩士論文系統

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研究生：	許文政 Wun-jheng Syu
論文名稱：	利用生物素改質之幾丁聚醣進行基因原位傳送 The Use of Biotinylated Chitosan for In-situ Gene Delivery
指導教授：	胡威文 Wei-wen Hu
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程與材料工程學系 Department of Chemical & Materials Engineering
畢業學年度：	99
語文別：	中文
論文頁數：	125
中文關鍵詞：	生物素改質、原位基因傳送、幾丁聚醣
外文關鍵詞：	Chitosan, in situ gene delivery, Biotinylation
相關次數：	點閱：8 下載：0
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本研究中將非病毒載體—幾丁聚醣以化學改質的方法，進行不同程度的
生物素接枝，並以正負電荷吸引之方式，將生物素幾丁聚醣與質體DNA，
以不同胺基/磷酸根莫耳比組裝成奈米粒子。經由動態雷射散射粒徑分佈儀
顯示，奈米粒子的粒徑大小分佈在66.8 nm 至109.8 nm 以及表面電位介於
21.6 至36.0 mV 之間，均適合應用於基因傳送。此外，經由生物素改質的
幾丁聚醣，依然能提供質體DNA 良好的包覆率與保護能力。同時由LDH
及MTS 測試顯示，生物素改質並不會影響生物合適性，甚至能減緩細胞毒
性。透過生物素與卵白素間作用力的調整，生物素之奈米粒子可以被固定
於基材表面進行基因原位傳送，相較於以懸浮方式送藥的未改質奈米粒
子，本方法可以提升約4.5 倍的轉染效率。

In this research, we modified chitosan, the non-viral vector, with different
degrees of biotin moieties. By electrostatic interaction, we prepared
self-assemble nanoparticles using biotinylated-chitosan and plasmid DNA in
different amine/phosphate ratio. The dynamic laser scattering experiment has
demonstrated that particle sizes of biotinylated nanoparticles were between the
66.8 nm to 109.8 nm and the Zeta potential was between 21.6 to 36.0 mV,
suggesting that these nanoparticles were suitable for gene delivery. In addition,
biotinylated chitosan may effectively bind DNA to protect it from DNase I
digestion. The LDH and MTS assays suggested that biotin modification did not
only maintain the biocompatibility of chitosan, but also reduce the cytotoxicity.
Through the optimatization of biotin-avidin interaction, biotinylated
nanoparticles may be immobilized on material surfaces for in situ gene delivery.
Compares to bolus gene delivery, our developed in situ gene delivery may
enhance the transfection efficiency of 4.5 folds. These results suggest that our
strategy successfully improved transfection efficiency of non-viral vectors.

目錄
摘要........................................................................................................................I
Abstract ................................................................................................................ II
致謝..................................................................................................................... III
目錄.....................................................................................................................IV
圖目錄.............................................................................................................. VIII
表目錄.................................................................................................................. X
縮寫表.................................................................................................................XI
第壹章 序論...................................................................................................... 1
第貳章 文獻回顧.............................................................................................. 3
2-1 組織工程................................................................................................ 3
2-2 基因傳送療法........................................................................................ 5
2-3 基因載體................................................................................................ 7
2-4 原位基因傳送...................................................................................... 10
V
2-4-1 幾丁聚醣應用於原位基因傳送.............................................. 10
2-4-2 生物素與卵白素作用力.......................................................... 11
2-5 實驗假設.............................................................................................. 12
第參章 實驗材料與方法................................................................................ 14
3-1 試藥與原料.......................................................................................... 14
3-1-1 質體DNA ................................................................................ 14
3-1-2 細胞.......................................................................................... 16
3-1-3 藥品.......................................................................................... 16
3-2 儀器...................................................................................................... 18
3-3 試藥配製.............................................................................................. 19
3-4 質體DNA 純化................................................................................... 21
3-5 HEK293T 細胞培養............................................................................. 24
3-6 幾丁聚醣之分子量對轉染效率影響................................................. 27
3-6-1 細胞轉染.................................................................................. 27
VI
3-6-2 螢光表現量分析...................................................................... 28
3-6-3 ONPG 分析............................................................................... 28
3-7 生物素改質之幾丁聚醣製備............................................................. 31
3-7-1 生物素改質(Biotinylation) ...................................................... 31
3-7-2 生物素接枝量分析.................................................................. 31
3-8 生物素之奈米粒子製備及物理化學性質鑑定................................. 33
3-8-1 生物素之奈米粒子製備.......................................................... 33
3-8-2 雷射粒徑分佈儀分析.............................................................. 33
3-8-3 掃描式電子顯微鏡觀察.......................................................... 33
3-8-4 包覆率分析.............................................................................. 34
3-8-5 保護能力分析.......................................................................... 35
3-8-6 MTS 測試對細胞活性分析...................................................... 36
3-8-7 乳酸脫氫酶(LDH)測試對細胞毒性分析............................... 37
3-9 原位基因轉染...................................................................................... 39
VII
第肆章 結果與討論........................................................................................ 43
4-1 幾丁聚醣之分子量對轉染效率影響................................................. 43
4-2 生物素改質之幾丁聚醣製備............................................................. 47
4-2-1 生物素接枝量分析.................................................................. 49
4-3 奈米粒子物理化學性質鑑定............................................................. 51
4-3-1 動態雷射散射粒徑分佈儀...................................................... 51
4-3-2 掃描式電子顯微鏡觀察.......................................................... 54
4-3-3 包覆率分析.............................................................................. 55
4-3-4 保護能力分析.......................................................................... 60
4-3-5 MTS 測試對細胞活性分析................................................... 63
4-3-6 乳酸脫氫酶(LDH)測試對細胞毒性分析............................... 65
4-4 原位基因轉染...................................................................................... 67
第伍章 結論.................................................................................................... 76
參考文獻............................................................................................................. 97
VIII
圖目錄
圖 2 - 1 幾丁聚醣 (Chitosan)分子結構圖...................................................... 11
圖 3 - 1 pCMV-β結構圖.................................................................................. 14
圖 3 - 2 pEGFP-C3 結構圖................................................................................ 15
圖 4 - 1 β-Gal assay 反應圖............................................................................ 44
圖 4 - 2 生物素改質反應圖............................................................................. 47
圖 4 - 3 HABA assay 示意圖............................................................................. 49
圖 4 - 4 MTS assay 反應圖............................................................................... 64
圖 4 - 5 不同分子量的幾丁聚醣對細胞之轉染效率(pEGFP 轉染).............. 76
圖 4 - 6 不同分子量的幾丁聚醣對細胞之轉染效率(pCMVβ轉染) ............. 78
圖 4 - 7 生物素接枝程度分析(HABA assay).................................................. 79
圖 4 - 8 表面電位及粒徑大小分析................................................................. 80
IX
圖 4 - 9 掃描式電子顯微鏡............................................................................. 82
圖 4 - 10 螢光標定法分析包覆率.................................................................... 83
圖 4 - 11 電泳法分析包覆率............................................................................. 84
圖 4 - 12 保護能力分析.................................................................................... 85
圖 4 - 13 細胞活性分析(MTS assay)................................................................ 89
圖 4 - 14 細胞毒性分析(LDH assay)................................................................ 90
圖 4 – 15 藉由調整卵白素鍍盤濃度來控制Biotin-NPs 的原位基因效率... 91
圖 4 - 16 不同卵白素鍍盤濃度對轉染效率的影響........................................ 95
圖 4 - 17 DNA 劑量對轉染效率影響............................................................... 96
X

                                

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