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系統識別號 U0007-1704200714554162
論文名稱(中文) 以嵌段式聚合微膠體投與大分子和小分子物質到裸鼠的不同組織
論文名稱(英文) Delivery of Large and Small Molecules to Different Tissues of Nude Mice by Polymeric Micelles (PM)
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 93
學期 2
出版年 94
研究生(中文) 劉哲亨
研究生(英文) Che-Heng Liu 
學號 m301092018
學位類別 碩士
語文別 中文
口試日期
論文頁數 0頁
口試委員 指導教授-廖嘉鴻
中文關鍵字 微膠體  裸鼠  藥物 
英文關鍵字 Polymeric Micelles  gene delivery  Methylprednisolone 
學科別分類
中文摘要 本研究的初步目標,在於以嵌段式聚合微膠體作為載體,包覆三種物質,第一種是具有非特異性啟動子質體:pCMV-LacZ,以每天投與三個口服劑量,一共兩天的方式,在口服投與六個劑量,四十八小時之後,其基因的表現可以在裸鼠脊髓組織經由X-gal定性染色的方式觀察得到。第二種是具有特異性啟動子質體:pKeratin 12-LacZ,以每天點眼液投與三個劑量,一共兩天的方式,在投與六個劑量,四十八小時之後,而其基因的表現可以在裸鼠眼睛的上皮組織組織有所表現。第三種是醣質類固醇藥物-methylprednisolone (MP), 以靜脈注射的方式投與,觀察在七小時之內signal transducers and activators of transcription 5a (STAT5a), growth associated protein-43 (GAP-43) 基因表現,結果可以觀察到在裸鼠脊髓組織內STAT5a和GAP43有被MP影響其效果。
英文摘要 The goal of this project was used polymeric micelles (PM) as a molecule carrier. Three types of molecules were selected. First, orally deliver a non-specificity promoter plasmid (pCMV-LacZ) (7.2Kb). There times a day with six doses, after 48 hours, the expression of LacZ gene was observed in the nude mouse spinal cord tissue by the X-gal qualitative measurement. Secondly, eye drops deliver a specificity cornea-epithelial promoter plasmid (pKeratin 12-LacZ) (7.4Kb). There times a day with six doses, after 48 hours, LacZ the gene was also observed in the nude mouse epithelial region tissue. Thirdly, intravenous delivery of methylprednisolone (MP) was observed the signal transducers and activators of transcription 5a (STAT5a) and growth associated protein-43 (GAP-43) gene expression with 7 hours. It was found that STAT5a and GAP-43 were modified by PM in the nude mouse spinal cord tissue.
論文目次 目錄 目錄---------------------------------------------------------------------------------I 附圖目錄-------------------------------------------------------------------------VI 中文摘要-----------------------------------------------------------------------VIII 英文摘要-------------------------------------------------------------------------IX 第一章 序論-----------------------------------------------------------------------1 第一節 嵌段式共聚合物--------------------------------------------------------1 第二節 基因治療與方法--------------------------------------------------------1 1.基因治療-------------------------------------------------------------------------1 2.基因載體-------------------------------------------------------------------------1 2.1病毒型載體------------------------------------------------------------------2 2.1.1反轉錄病毒載體-------------------------------------------------------2 2.1.2 Lentivirus ---------------------------------------------------------------2 2.1.3腺病毒載體------------------------------------------------------------3 2.1.4腺相關病毒載體-------------------------------------------------------3 2.2非病毒型載體---------------------------------------------------------------4 2.2.1直接注射----------------------------------------------------------------4 2.2.2微脂粒-------------------------------------------------------------------4 2.2.3高分子聚合物----------------------------------------------------------5 2.2.4物理方法---------------------------------------------------------------6 第三節 細胞專一性的基因傳遞----------------------------------------------6 1.投與路徑的選擇-------------------------------------------------------------6 2.載體或是配位基-------------------------------------------------------------7 3.組織或細胞專一性的啟動子----------------------------------------------7 4.專一性啟動因子-Keratins(角蛋白)-------------------------------------7 5.眼睛中角膜的構造----------------------------------------------------------7 第四節 醣質類固醇藥與訊息傳遞 ------------------------------------------9 1.醣質類固醇藥物-------------------------------------------------------------9 2.信號傳導和轉錄?動因數5a --------------------------------------------9 3.生長相關的蛋白質43-----------------------------------------------------11 第二章 研究目的---------------------------------------------------------------12 第三章 研究方法與步驟------------------------------------------------------13 第一節 質體 DNA---------------------------------------------------------------13 1.質體 DNA的種類---------------------------------------------------------13 2.質體DNA之大量製備----------------------------------------------------13 第二節 聚合微膠體溶液------------------------------------------------------14 1.聚合微膠體之形成(臨界微膠濃度)------------------------------------14 2.聚合微膠體溶液之製備--------------------------------------------------14 3.聚合微膠體溶液之物化性質---------------------------------------------15 聚合微膠體溶液之表面電位(zeta potential)與粒徑大小----------15 4.DNA-聚合微膠體溶液之安定性----------------------------------------15 4.1聚合微膠體口服液----------------------------------------------------15 4.2聚合微膠體點眼液----------------------------------------------------15 第三節 體內基因傳遞藥物的傳遞------------------------------------------16 1.裸鼠(BALB/c-nu)------------------------------------------------------16 第四節 蛋白質濃度之測定---------------------------------------------------16 1.標準曲線的配製-----------------------------------------------------------16 2.檢品分析流程--------------------------------------------------------------17 第五節 b-galactosidase 的定量分析----------------------------------------17 1.標準曲線的配製-----------------------------------------------------------17 2.檢品分析流程--------------------------------------------------------------18 第六節 b-galactosidase之定性分析-----------------------------------------18 1.實驗流程--------------------------------------------------------------------19 第七節 反轉錄-聚合酵素鏈鎖反應-----------------------------------------19 1.組織RNA之萃取----------------------------------------------------------19 2.第一股cDNA的製備------------------------------------------------------21 2.1 b-actin的check-------------------------------------------------------21 2.2 b-gal--------------------------------------------------------------------22 第八節 膠體電泳位移分析---------------------------------------------------23 1.細胞核蛋白的製備--------------------------------------------------------23 2.DNA探針的序列-----------------------------------------------------------23 3.探針的標定-----------------------------------------------------------------24 4.膠體電泳位移分析--------------------------------------------------------24 第九節 即時聚合?連鎖反應法---------------------------------------------25 1. SYBR-system -------------------------------------------------------------25 1.1實驗流程---------------------------------------------------------------25 2. TaqMan system------------------------------------------------------------26 2.1實驗流程 -------------------------------------------------------------27 附圖--------------------------------------------------------------------------------28 第四章 研究成果與討論------------------------------------------------------33 第一節 聚合微膠體溶液之表面電位大小---------------------------------33 1.聚合微膠體口服液--------------------------------------------------------33 2.聚合微膠體點眼液--------------------------------------------------------33 3.聚合微膠體注射液--------------------------------------------------------33 第二節 聚合微膠體溶液之粒徑大小---------------------------------------34 1.聚合微膠體口服液--------------------------------------------------------34 2.聚合微膠體點眼液--------------------------------------------------------34 3.聚合微膠體注射液--------------------------------------------------------34 第三節 臨界微膠濃度的測定------------------------------------------------35 第四節 DNase I安定性分析--------------------------------------------------35 1.聚合微膠體口服液--------------------------------------------------------35 2.聚合微膠體點眼液--------------------------------------------------------35 第五節 b-galactosidase 的定量分析----------------------------------------36 1.裸鼠脊隨組織b-galactosidase 的定量分析---------------------------36 2.裸鼠眼球組織b-galactosidase 的定量分析---------------------------36 第六節 b-galactosidase之定性分析-----------------------------------------37 1裸鼠脊隨組織b-galactosidase 的定性分析----------------------------37 第七節 膠體電泳位移分析---------------------------------------------------37 第八節 即時聚合?連鎖反應法之分析(real time Polymerase Chain Reaction) --------------------------------------------------------------38 第五章 結論---------------------------------------------------------------------39 附圖--------------------------------------------------------------------------------41 第六章 參考文獻---------------------------------------------------------------50
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