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研究生:林巾妤
研究生(外文):LIN,JIN-YU
論文名稱:細胞穿膜胜肽GLA、GYR與攜帶藥物穿透血腦障壁的機制探討
論文名稱(外文):Studies on the blood brain barrier penetrating mechanism of GLA and GYR peptide-drug conjugates
指導教授:吳雨珊吳雨珊引用關係
指導教授(外文):WU,YU-SHAN
口試委員:龍鳳娣謝佳宏
口試委員(外文):LONG,FENG-DIHSIEH,CHIA-HUNG
口試日期:2015-07-07
學位類別:碩士
校院名稱:東海大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:88
中文關鍵詞:細胞穿透性胜肽FITC
外文關鍵詞:Cell penetrating peptideFITC
相關次數:
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  • 點閱點閱:104
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血腦障壁(Blood-brain barrier,簡稱BBB)是由多層內皮細胞緊密連接而成,用來保護大腦不受血液中外來物質的傷害並維持腦中環境的平衡,因而使的多數有效的藥物無法穿透血腦障壁到達作用位置以達到治療效果。為了解決上述問題,我們選用Van Rooy et al. 提出以噬菌體展示技術(phage display) 方法找到的兩個胜肽序列GLA、GYR作為載體,並選用NMDA酵素中的glycine site的拮抗劑 ”1,4-Dihydroquinoxaline-2,3-diones” 類型化合物作為模型藥物,此類型藥物無法獨自穿越血腦障壁,再將胜肽與藥物做連接以進行穿透BBB的研究,並進而探討胜肽穿膜的機制。本研究確認GLA(N)連接模型藥物時可穿透BBB,且確認GLA與GYR並不以直接易位進入細胞內。
The blood–brain barrier is formed by brain endothelial cells, which are connected by tight junctions. It can protect the brain from foreign substances damage and maintain the balance of the environment. However, it makes a lot of effective drugs can not penetrate the blood-brain barrier to reach the active position. So the drugs can not achieve therapeutic effect. To deal with this problem, we chose Van Rooy et al. reported the use of phage display to find GLA, GYR to be peptide carrier, and select a model drug which binding glycine site’s NMDAR “1,4-Dihydroquinoxaline-2,3- diones” analogue, this type drug can not penetrate BBB along. So we conjugate drug to the peptide to test if it can penetrate BBB or not, and study on the blood brain barrier penetrating mechanism of peptide. In this study, GLA(N) conjugate the drug can penetrate BBB, and GLA, GYR across into cell not use direct translocation.
目錄
中文摘要………………………………………………………...…I
英文摘要………………………………………………………..…II
圖目錄…………………………………………………………..…V
表目錄………………………………………………………….. VIII
附錄……………………………………………………………..…IX
第一章 緒論……………………………………………………..…1
1.1. 細胞穿膜胜肽(CPPs)簡介…………………………..…2
1.2. 細胞穿膜胜肽的跨膜模式及機制…………………..…3
1.3. 血腦障壁(BBB) ……………………………………...…9
1.4. 實驗設計與研究………………………………………11
第二章 實驗與方法………………………………………………15
2.1. 胜肽之設計與合成………………………….……….22
2.1.1 固相胜肽合成法……………………………..…22
2.1.2 胜肽與藥物的合成…………………………..…25
2.1.3 茚三酮測試………………………………..……27
2.1.4 逆相高效能液相層析法……………………..…28
2.1.5 基質輔助雷射脫附游離飛行時間質譜儀…..…29
2.1.6 合成含螢光染劑FITC的胜肽………………30
2.2. 體外穿膜機制實驗……………………………………32
2.2.1 細胞培養……………………………………..…32
2.2.2 平行人工滲透模型測試……………………..…34
2.2.3 體外穿膜機制實驗…………………………..…35
2.3. 血清穩定性實驗………………………………………36
2.4. 體內動物穿膜實驗……………………………………37
2.4.1 放射線125I標定…………………………………37
2.4.2 體內動物穿膜實驗…………………………..…38
第三章 結果與討論………………………………………………39
3.1. 胜肽與FITC合成……………………..………………39
3.2. 體外穿膜機制實驗結果………………………………45
3.3. 血清穩定性實驗結果…………………………………53
3.4. 體內動物穿膜實驗結果………………………………56
第四章 結論………………………………………………………59
第五章 未來展望…………………………………………………60

圖目錄
圖1-1. CCPs穿透細胞膜機制…………………………………..…4
圖1-2. 左邊為超環狀孔洞示意圖、中間為地毯式包覆示意圖、
右邊為桶狀式穿鑿孔洞示意圖.……,………………….…6
圖1-3. 逆微胞示意圖…………………………………...…………7
圖1-4. 血腦障壁(BBB)的組成 ……..…..……………….………10
圖1-5. 通過血腦障壁(BBB)的機制…………………….…….….11
圖1-6. (A)回收經由Glycine清洗後的噬菌體(與細胞表面結合
的噬菌體)
(B)細胞裂解後回收噬菌體(細胞內部的噬菌體)..........…12
圖1-7. (A)將GLA、GYR、GTW與控制組YLR培養到
hCMEC/D3與HUVEC細胞上,將穿透過細胞的噬菌
體回收(細胞表面的噬菌體),並以百分比表示穿透力
(B)細胞裂解後回收噬菌體(細胞內部的噬菌體),並以百
分比表示穿透力………...……………………………..12
圖1-8. 胜肽GLA經由靜脈注射後的生物分布圖………………13
圖1-9. 6,7-dichloro-5-nitroquin-oxaline-2,3(1H,4H)-dione
衍生物………………………...………………………......14
圖2-1. 固相胜肽合成流程圖………………...………………..…23
圖2-2. Piperidine切除胺基酸N端上的Fmoc保護基之反應機
構……….….………..…..…..………………....………….25
圖2-3. NMDAR的拮抗劑及其衍生物……....…………….……..26
圖2-4. 茚三酮與胺基酸的反應機構……………………….……27
圖2-5. 為胜肽與FITC合成反應機構…………………...………31
圖2-6. PAMPA模型………………………………………………34
圖3-1. FITC-GLA -NH2的MALDI-TOF-MS質譜圖…….……41
圖3-2. FITC與最後一個胺基酸環化機制……………...…….…41
圖3-3. FITC-Ahx-GLA-K的MALDI-TOF-MS質譜圖..........…42
圖3-4. FITC-Ahx-GYR的MALDI-TOF-MS質譜圖...………...42
圖3-5. FITC-GLA -NH2螢光儀檢測結果………………………43
圖3-6. Fmoc-K(FITC)-NH2的MALDI-TOF-MS質譜圖............44
圖3-7. Fmoc-FARDFVAK-(FITC)-NH2的MALDI-TOF-MS質
譜圖…………………..........................................................45
圖3-8. ELISA測得FITC-GLA的螢光量……………………….48
圖3-9. ELISA測得FITC-GYR的螢光量……………….………48
圖3-10. ELISA測得FITC與FITC-GYR(C)的螢光量……...…49
圖3-11. 螢光顯微鏡下的FITC與FITC-GYR(C)………….…..49
圖3-12. 流式細胞儀測得FITC與FITC-GYR(C)的螢光
量……...……………………………...…………………50
圖3-13. 流式細胞儀測得FITC-GLA的螢光量百分比……...…51
圖3-14. 流式細胞儀測得FITC-GYR的螢光量百分比………...52
圖3-15. 流式細胞儀測得FITC-peptide的螢光量…...…………53
圖3-16. 羧酸型(C)與醯胺型(N)的GLA在不同時間點中的血清
穩定性…………………………………..………………55
圖3-17. 羧酸型(C)與醯胺型(N)的GYR在不同時間點中的血清
穩定性………………………………………………..…55
圖3-18. 放射線分別在小鼠的血液及大腦中的量……………...57
圖3-19. 小鼠的血液與大腦內的放射線比……………………...58

表目錄
表2-1. 實驗材料、用途以及購買來源…….…………..…………17
表2-2. 欲合成的胜肽及胜肽藥物…….…………………………26
表2-3. 欲合成含FITC的胜肽序列…….…………………..……31
表3-1.合成胜肽與胜肽藥物複合物之分子量…………….…..…39
表3-2. PAMPA穿膜實驗結果……………………………………46
表3-3. 胜肽序列經由RP-HPLC定量結果……………………...54
表3-4. 確認125I是否有與藥物連接上………………………….56

附錄
附錄一、 GLA(N) 之 RP-HPLC 層析圖………………………67
附錄二、 FITC-GLA(N) 之 RP-HPLC 層析圖……………….68
附錄三、 GLA(C) 之 RP-HPLC 層析圖………………………69
附錄四、 FITC-GLA(C) 之 RP-HPLC 層析圖…………….…70
附錄五、 GYR(N) 之 RP-HPLC 層析圖………………………71
附錄六、 FITC-GYR(N) 之 RP-HPLC 層析圖……………….72
附錄七、 GYR(C) 之 RP-HPLC 層析圖………………………73
附錄八、 FITC-GYR(C) 之 RP-HPLC 層析圖…………….…74
附錄九、 433-6C-GLA(N) 之 RP-HPLC 層析圖……………..75
附錄十、 Tat-Y(C) 之 RP-HPLC 層析圖……………………...76
附錄十一、 GLA(N) 之純化後的 MALDI-TOF MS質譜圖….77
附錄十二、 FITC-GLA(N) 之純化後的 MALDI-TOF MS質譜
圖……………………………………………………77
附錄十三、 GLA(C) 之純化後的 MALDI-TOF MS質譜圖….78
附錄十四、 FITC-GLA(C) 之純化後的 MALDI-TOF MS質譜
圖……………………………………………………78
附錄十五、 GYR(N) 之純化後的 MALDI-TOF MS質譜圖….79
附錄十六、 FITC-GYR(N) 之純化後的 MALDI-TOF MS質譜
圖……………………………………………………79
附錄十七、 GYR(C) 之純化後的 MALDI-TOF MS質譜圖….80
附錄十八、 FITC-GYR(C) 之純化後的 MALDI-TOF MS質譜
圖……………………………………………………80
附錄十九、 433-6C-GLA(N) 之純化後的 MALDI-TOF MS質
譜圖………………………………………………....81
附錄二十、 Tat-Y(C) 之純化後的 MALDI-TOF MS質譜圖…81
附錄二十一、 PAMPA原始數據……………………………..…..…82
附錄二十二、 血清穩定性的原始數據…………………….….…82
附錄二十三、 體內動物穿膜實驗的原始數據…………………..83
附錄二十四、 流式細胞儀FITC原始圖譜…………. …………..84
附錄二十五、 流式細胞儀FITC-GLA(N)及其抑制劑
之原始圖譜……,,,,……. ………………..………85
附錄二十六、 流式細胞儀FITC-GLA(C)及其抑制劑
之原始圖譜…………. …………………………..86
附錄二十七、 流式細胞儀FITC-GYR(N)及其抑制劑
之原始圖譜…………. ………………………..…87
附錄二十八、 流式細胞儀FITC-GYR(C)及其抑制劑
之原始圖譜…………. …………………………..88

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