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系統識別號 U0007-2607201110282100
論文名稱(中文) 以高效能液相層析法應用於質體 DNA與聚合微膠混合體的測定
論文名稱(英文) Determination of Plasmid DNA/Polymeric Micelles Complexes Using High-Performance Liquid Chromatography
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 99
學期 2
出版年 100
研究生(中文) 李世翔
研究生(英文) Shih-Hsiang Lee
學號 M301098033
學位類別 碩士
語文別 中文
口試日期 2011-06-18
論文頁數 65頁
口試委員 指導教授-廖嘉鴻
委員-高純琇
委員-邱士娟
中文關鍵字 質體DNA  聚合物  高效能液相層析法 
英文關鍵字 plasmid  polymer  HPLC 
學科別分類
中文摘要 先前本實驗室曾使用此三嵌段共聚合物當作質體 DNA的載體,證實可以增加轉殖基因的表現量,然而實際質體 DNA的包覆率 (encapsulation efficiency) 仍不清楚,因此本實驗目的為探討在使用微膠體當作載體包覆質體 DNA 後的包覆率。首先利用Zeta potential、 pH與膠體電泳分析對質體 DNA、聚合微膠體溶液、質體 DNA與聚合微膠混合體溶液三者的物理特性做探討,質體 DNA為對 pH 敏感的物質,而聚合微膠體溶液則相當穩定不會隨著 pH值改變,質體 DNA與聚合微膠混合體溶液則有兩種 Zeta 電位的分布;另外在膠體電泳分析實驗中,在應用聚合微膠體包覆質體 DNA後,有不同凝集 (aggregation) 現象出現。利用 HPLC 配合在 UV 260 nm偵測下,質體 DNA溶液在第9.3分有單一 peak形成,而在質體 DNA與聚合微膠混合體溶液,則在第6.3 分鐘中另一新形成之 peak,同時分別收集質體 DNA和質體 DNA與聚合微膠混合體在 HPLC分析下所形成 peak範圍,之後利用加入螢光染劑 Picogreen®、膠體電泳分析和即時定量聚合酶鏈鎖反應等方法對其定性及定量,做再次確認評估。
在利用 HPLC分析之圖譜計算包覆率後,經0.3%聚合微膠體包覆1.5、4.5、9 ng/μl質體 DNA後,其包覆量分別為1303.9 ± 139.75 ng、2541.7 ± 621.22 ng、2639.4 ± 555.08 ng,包覆率則各為37.76 ± 3.90%、23.96 ± 6.00%、13.62 ± 2.83%,與利用即時定量聚合酶鏈鎖反應所得包覆率各為30.97 ± 8.02、22.31 ± 8.97、13.77± 1.68 %,兩者結果類似;而經3%聚合微膠體包覆1.5、4.5、9 ng/μl質體 DNA後,其包覆量分別為1558.8 ± 86.75 ng、3561.2 ± 247.32 ng、4215.2 ± 356.67 ng,包覆率則各為44.84 ± 2.10%、33.71 ± 2.50%、22.09 ± 1.82%。而在藥物釋放過程中,質體 DNA與聚合微膠混合體 (10 ng/μl plasmid DNA/0.3%、3% polymeric micelles)的釋放速率為7.39、8.19 ng/min。
英文摘要 Previously used the triblock copolymer as a carrier encapsulated of plasmid DNA, it could effectively enhance the gene expression. However, encapsulation of plasmid DNA still remains unsolved. The purpose of this study is determination encapsulation efficiency of plasmid DNA with polymeric micelles. Characterizations of plasmid DNA、polymeric micelles and plasmid DNA with polymeric micelles solutions by Zeta potential, pH effect and gel electrophoresis. The results showed that plasmid DNA was pH-sensitive with decreasing Zeta potential value, and plasmid DNA with polymeric micelles showed two Zeta potential peaks distribution, however polymeric micelles was stable under pH changed. Using electrophoresis observation, after application of polymeric micelles encapsulated plasmid DNA, the different aggregation were observed. Under High-Performance Liquid Chromatography and UV-VIS detector, the plasmid DNA was observed at 9.3 minutes, and plasmid DNA with polymeric micelles solutions had observed two peaks peak at 6.3 and 9.3 minutes. In addition, the fractions of these two peaks of plasmid DNA and plasmid DNA with polymeric micelles were consistently confirmed by Picogreen®, gel electrophoresis, and QPCR evaluation.
The three different plasmid DNA (1.5,4.5,9 ng/μl) with 0.3% polymeric micelles were found the encapsulated amount were 1303.9 ± 139.75 ng、2541.7 ± 621.22 ng、2639.4 ± 555.08 ng, respectively. The encapsulation efficiency were 37.76 ± 3.90%、23.96 ± 6.00%、13.62 ± 2.83%, respectively by HPLC analysis. When increasing polymeric micelles to 3% with 1.5,4.5,9 ng/ μl plasmid DNA, encapsulation were 1558.8 ± 86.75 ng、3561.2 ± 247.32 ng、4215.2 ± 356.67 ng, respectively. The encapsulation efficiency were 44.84 ± 2.10%、33.71 ± 2.50%、22.09 ± 1.82%, respectively. Furthermore, the in vitro release rates of 10 ng/μl plasmid with 0.3% 、3% polymeric micelles were 7.39、8.19 ng/ μl, respectively.
論文目次 目錄 I
附表目錄 IV
附圖目錄 V
中文摘要 VIII
Abstract IX
第一章 緒論 1
第一節 三嵌段共聚合物 (triblock coploymer) 1
第二節 小分子藥物上的應用 2
第三節 大分子藥物上的應用 4
第四節 DNA的定量 (Quantitation of DNA) 6
4.1. O.D.260 nm 測定法 10
4.2. 螢光染劑法 10
4.3. 即時定量聚合酶鏈鎖反應 12
(Quantitative real time polymerase chain reaction, QPCR) 12
4.4. 高效能液相層析法 14
(High-Performance LiquidChromatography, HPLC) 14
4.5. 液相層析串聯式質譜儀 15
(Liquid Chromatograph/Mass Spectrometer, LC/MS) 15
第五節 DNA包覆率的計算 16
第二章 研究目的 23
第三章 實驗材料與方法 24
第一節 實驗材料與儀器 24
1.1. 試藥 24
1.2. 儀器、耗材 24
1.3. 質體 DNA製備 25
1.4. 引子 (primer) 序列 26
1.5. 三嵌段式共聚合物 26
第二節 實驗方法 27
2.1. 質體 DNA物化性質之探討 27
2.2. 三嵌段共聚合物 PEO-PPO-PEO物化性質之探討 28
2.3. 質體/聚合微膠體 (Plasmid/ Polymeric micelles, P/PM) 之物化特性探討 29
2.4. 高效能液相層析 (HPLC) 分析方法之建立 31
2.5. 質體 DNA (Plasmid) 及質體/聚合微膠體 (P/PM) 之確認 33
2.6. 包覆率的評估 35
2.7. P/PM的釋放曲線 36
第四章 實驗結果與討論 38
第一節 質體 DNA物化性質之探討 38
1.1. pH值對質體 DNA的影響 38
1.2. 不同濃度質體 DNA之膠體電泳分析 40
第二節 三嵌段共聚合物 PEO-PPO-PEO物化性質之探討 41
2.1. 聚合微膠體之臨界微膠體濃度之測量 41
2.2. pH 值對聚合微膠體的影響 42
第三節 質體/聚合微膠體 (Plasmid/Polymeric micelles, P/PM) 之物化特性探討 45
3.1. 不同濃度質體/聚合微膠體之膠體電泳分析 45
3.2. pH 值對P/PM的影響 46
第四節 高效能液相層析 (HPLC) 分析方法之建立與確效 47
4.1. 質體 DNA之標準檢量線建立 47
4.2. 質體 DNA分析方法之確效 (Validation) 49
4.3. 質體/聚合微膠體 (Plasmid/ Polymeric micelles, P/PM) 之分析 50
第五節 質體 DNA (Plasmid) 及質體/聚合微膠體 (P/PM) 之確認 51
5.1. 收集 HPLC 二個波峰範圍進行膠體電泳分析 51
5.2. 收集 HPLC 二個波峰範圍進行 QPCR 絕對定量 52
5.3. 質體 DNA加入 Quant-iT™ Picogreen® 55
第六節 包覆率之評估 57
6.1. 不同質體 DNA濃度包覆率之比較 57
6.2. 不同聚合微膠體濃度包覆率之比較 58
第七節 P/PM的釋放曲線 59
第五章 結論 61
第六章 參考文獻 62

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