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研究生:嚴國城
研究生(外文):Kuo Chen Yen
論文名稱:放射性標幟AnnexinV衍生物的放射化學與生物特性研究
論文名稱(外文):Radiochemical and biological characterizations of radiolabeled Annexin V derivatives
指導教授:魏孝萍
指導教授(外文):S. P. Wey
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學物理暨影像科學研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:116
中文關鍵詞:細胞凋亡Annexin V衍生蛋白磷酯基絲胺酸A6L15 融合蛋白
外文關鍵詞:apoptosisAnnexin V derivativesphosphatidylserineA6L15 fusion protein
相關次數:
  • 被引用被引用:1
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  • 下載下載:25
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細胞凋亡 (apoptosis) 是細胞程序性死亡 (program cell death;
PCD) 的一種,是生物體內自我調節的機制之一,對正常細胞的成
長、發育及免疫機能的調節扮演重要的角色。Annexin V 是生物體內
固有的蛋白質,廣泛分布於胎盤、內皮細胞、腎臟、心肌、骨骼肌、
紅血球、血小板等組織或細胞內,與磷酯基絲胺酸 (phosphatidylserine;
PS)具有高度親和力 (Kd = 10-9M),而PS 的細胞膜外翻為細胞凋亡一
項重要的特色,因此,可以根據此特異性利用Annexin V 偵測細胞凋
亡。本研究包括兩大部份:(1) 評估鎝-99m 標幟Annexin V C 端內配
位基衍生蛋白應用於細胞凋亡造影的可行性。(2) 評估ghost 紅血球
細胞用於放射性標幟Annexin V 體外活性分析之可行性。放射薄層色
層分析及size-exclusion HPLC 分析都顯示含C端內配位基衍生蛋白之
鎝-99m-AV-47R 具有相當高的放射化學純度,且與紅血球細胞的結合
比率較含N 端內配位基衍生蛋白之鎝-99m-AV-128 高。此外,本研究
也證實ghost 紅血球細胞是一個穩定的分析模型用以評估放射性標幟
Annexin V 的體外生物活性;放射性碘標幟A6L15 融合蛋白可能比
Annexin V 更適合用於細胞凋亡的偵測。
Apoptosis is a kind of programmed cell death and a self-modulation mechanism of organism. It plays an important role for cellular development and immune modulation. Annexin V has a high affinity to bind phosphatidylserine (PS) and intersperses among placenta, kidney, cardial muscle, skeletal muscle, erythrocyte and other tissues and cells . Since PS externalization is an important feature of apoptosis, so Annexin V has been employed to detect apoptosis. This study aimed: (1) to evaluate the feasibility of using 99mTc-labeled Annexin V mutant with
C-terminal endogenous ligand for imaging apoptosis; (2) to evaluate the feasibility of using ghost erythrocytes for assessing the binding affinities of radiolabeled Annexin V derivatives. In the first part, the results of radio-thin layer chromatography and size-exclusion HPLC revealed
99mTc-AV-47R with the best radiochemical purity, and the 99mTc-labeled Annexin V mutant with C-terminal extension showed higher erythrocyte binding bioactivity than those of N-terminal extension. In the second part, the ghost erythrocyte was demonstrated as a stable model for assessing
the in vitro biological affinity of radiolabeled Annexin V. The binding affinity of 125I-A6L15 fusion was better than 125I-Annexin V. Radioiodinated A6L15 fusion protein may be more suitable to decelop radiotracer for apoptosis detection.
目錄
誌謝........................................................................................................i
中文摘要............................................................................................. iii
英文摘要..............................................................................................iv
目錄.......................................................................................................v
圖目錄..................................................................................................ix
表目錄............................................................................................... xiii
第一章 序言及文獻探討……………………………………….. ..- 1 -
1-1 Annexin V 蛋白質.................................................................... - 1 -
1-2 何謂細胞凋亡......................................................................... - 2 -
1-2-1 細胞凋亡的定義........................................................... - 2 -
1-2-2 細胞凋亡的生理意義................................................... - 2 -
1-2-3 細胞凋亡的機制........................................................... - 4 -
1-3 體外偵測細胞凋亡的方法..................................................... - 5 -
1-3-1 TUNEL assay ................................................................. - 5 -
1-3-2 活化caspase 酵素分析................................................. - 6 -
1-3-3 DNA 凝膠電泳分析...................................................... - 6 -
1-3-4 螢光標定Annexin V .................................................... - 6 -
1-4 體內偵測細胞凋亡的方法..................................................... - 7 -
vi
1-5 放射性標幟Annexin V 的臨床應用..................................... - 8 -
1-5-1 心血管疾病................................................................... - 8 -
1-5-2 神經退化性疾病........................................................... - 8 -
1-5-3 腫瘤造影....................................................................... - 8 -
1-5-4 腫瘤治療療效評估....................................................... - 9 -
1-6 標幟Annexin V 的放射性核種........................................... - 10 -
1-6-1 氟-18 標幟Annexin V................................................ - 10 -
1-6-2 銦-111 標幟Annexin V .............................................. - 10 -
1-6-3 放射性碘標幟Annexin V .......................................... - 10 -
1-6-4 鎝-99m 標幟Annexin V............................................. - 11 -
1-7 論文主旨............................................................................... - 14 -
第二章 材料與方法......................................................................- 15 -
2.1 材料......................................................................................... - 15 -
2.2 儀器及設備............................................................................ - 17 -
2.3 實驗一:評估鎝-99m 標幟Annexin V C 端衍生蛋白應用於細
胞凋亡造影的可行性.................................................................. - 18 -
2-3-1 Annexin V 衍生蛋白................................................... - 18 -
2-3-2 Size-exclusion HPLC 分析Annexin V 衍生蛋白...... - 18 -
2-3-3 鎝-99m-Annexin V 衍生蛋白製備與放射化學分析- 19 -
vii
2-3-4 鎝-99m-Annexin V 衍生蛋白體外生物活性分析.... - 22 -
2-3-5 鎝-99m-Annexin V 衍生蛋白小鼠活體microSPECT 造影
分析........................................................................................ - 24 -
2-4 實驗二:評估Ghost 紅血球細胞用於放射性標幟Annexin V
體外活性分析之可行性.............................................................. - 26 -
2-4-1 Ghost 紅血球細胞製備............................................... - 26 -
2-4-2 A6L15 融合蛋白.......................................................... - 27 -
2-4-3 碘-125 標幟Annexin V 及A6L15 融合蛋白的製備與放
射化學分析............................................................................ - 28 -
2-4-4 Ghost 紅血球細胞分析碘-125 標幟Annexin V 及A6L15
融合蛋白體外生物活性實驗............................................... - 31 -
第三章 結果..................................................................................- 33 -
3-1 實驗一:評估鎝-99m 標幟Annexin V C 端衍生蛋白應用於細
胞凋亡造影的可行性.................................................................. - 33 -
3-1-1 Size-exclusion HPLC 分析Annexin V 衍生蛋白...... - 33 -
3-1-2 鎝-99m-Annexin V 衍生蛋白的標幟效率與純度分析….
................................................................................................ - 33 -
3-1-3 鎝-99m-Annexin V 衍生蛋白體外生物活性分析.... - 35 -
3-1-4 鎝-99m-Annexin V 衍生蛋白活體內microSPECT 造影分
viii
析............................................................................................ - 36 -
3-2 實驗二:評估Ghost 紅血球細胞用於放射性標幟Annexin V
體外活性分析之可行性.............................................................. - 37 -
3-2-1 Size-exclusion HPLC 分析Annexin V 和A6L15 融合蛋
白............................................................................................ - 37 -
3-2-2 碘-125 標幟Annexin V 及A6L15 融合蛋白的標幟效率
與純度分析............................................................................ - 37 -
3-2-2-3 碘-125 標幟Annexin V 及A6L15 融合蛋白冷藏穩定度
試驗........................................................................................ - 39 -
3-2-3 碘-125 標幟Annexin V 及A6L15 融合蛋白與ghost 紅血
球細胞結合的體外生物活性分析....................................... - 40 -
第四章 討論..................................................................................- 43 -
4-1 實驗一:評估鎝-99m 標幟Annexin V C 端衍生蛋白應用於細
胞凋亡造影的可行性.................................................................- 43 -
4-2 實驗二:評估Ghost 紅血球細胞用於放射性標幟Annexin V
體外活性分析之可行性.............................................................. - 47 -
第五章 結論..................................................................................- 50 -
參考文獻........................................................................................- 51 -
ix
圖目錄
圖一、Annexin V 晶體結構............................................................... - 55 -
圖二、細胞壞死與凋亡的型態區別................................................. - 56 -
圖三、細胞凋亡程序示意圖............................................................. - 57 -
圖四、以冷凍乾燥機製備Sn (Ⅱ)-Glucoheptonate 凍晶小瓶........ - 58 -
圖五、BIOSCAN AR2000 放射薄層色層分析儀。........................ - 59 -
圖六、Wallac Wizard 1470 加馬活度計數器.................................... - 60 -
圖七、Annexin V-KPI 融合蛋白與PS 結合的示意圖.................... - 61 -
圖八、AV-47 HPLC 分析紫外光 280 nm 吸收圖譜....................... - 62 -
圖九、AV-47R HPLC 分析紫外光 280 nm 吸收圖譜..................... - 63 -
圖十、AV-3 HPLC 分析紫外光 280 nm 吸收圖譜.......................... - 64 -
圖十一、AV-128 HPLC 分析紫外光 280 nm 吸收圖譜……………- 65 -
圖十二、鎝-99m-Annexin V 衍生蛋白ITLC-SG 放射薄層色層分析圖
譜......................................................................................... - 66 -
圖十三、鎝-99m-AV-47R 純化前的ITLC-SG 放射薄層色層分析圖譜.
............................................................................................. - 67 -
圖十四、鎝-99m-AV-47R 純化後的ITLC-SG 放射薄層色層分析圖譜.
............................................................................................. - 68 -
x
圖十五、鎝-99m-AV-47 size-exclusion HPLC 放射分析圖譜......... - 69 -
圖十六、鎝-99m-AV-47R size-exclusion HPLC 放射分析圖譜...... - 70 -
圖十七、鎝-99m-AV-3 size-exclusion HPLC 放射分析圖譜........... - 71 -
圖十八、鎝-99m-AV-128 size-exclusion HPLC 放射分析圖譜....... - 72 -
圖十九、未接受Doxorubicin 治療之CT-26 荷腫瘤BALB/c 小鼠 (對
照組) 注射鎝-99m 標幟AV-47R 後15 分鐘之下腹部
microSPECT / microCT 造影圖......................................... - 73 -
圖二十、未接受Doxorubicin 治療之CT-26 荷腫瘤BALB/c 小鼠 (對
照組) 注射鎝-99m 標幟AV-47R 後90 分鐘之全身
microSPECT / microCT 造影圖......................................... - 74 -
圖二十一、接受Doxorubicin 治療之CT-26 荷腫瘤BALB/c 小鼠注射
鎝-99m 標幟AV-47R 後15 分鐘之下腹部microSPECT /
microCT 造影圖.............................................................. - 75 -
圖二十二、接受Doxorubicin 治療之CT-26 荷腫瘤BALB/c 小鼠注射
鎝-99m 標幟AV-47R 後90 分鐘之全身部microSPECT /
microCT 造影圖.............................................................. - 76 -
圖二十三、Annexin V HPLC 分析紫外光 280 nm 吸收圖譜........ - 77 -
圖二十四、A6L15 融合蛋白 HPLC 分析紫外光 280 nm 吸收圖譜….
....................................................................................... - 78 -
xi
圖二十五、碘-125 離子之ITLC-SG 放射薄層色層分析圖譜。... - 79 -
圖二十六、碘-125-Annexin V 純化前之ITLC-SG 放射薄層色層分析圖
譜..................................................................................... - 80 -
圖二十七、碘-125-A6L15 融合蛋白純化前之ITLC-SG 放射薄層色層
分析圖譜......................................................................... - 81 -
圖二十八、碘-125-Annexin V 純化後之ITLC-SG 放射薄層色層分析圖
譜..................................................................................... - 82 -
圖二十九、碘-125-A6L15 融合蛋白純化後之ITLC-SG 放射薄層色層
分析圖譜......................................................................... - 83 -
圖三十、碘-125-Annexin V size-exclusion HPLC 放射分析圖譜... - 84 -
圖三十一、碘-125-A6L15 融合蛋白 size-exclusion HPLC 放射分析圖
譜。……………………………………………………..- 85 -
圖三十二、碘-125-Annexin V 保存在PBS 溶液 (pH = 7.4) 且冷藏於4
℃下的放射化學穩定度................................................. - 86 -
圖三十三、碘-125-A6L15 融合蛋白保存在PBS 溶液 (pH = 7.4) 且冷
藏於4 ℃下的放射化學穩定度..................................... - 87 -
圖三十四、反應時間對碘-125-Annexin V 及碘-125-A6L15 融合蛋白
與未稀釋之ghost 紅血球細胞的結合比率的影響 (n = 3)..
........................................................................................ .- 88 -
xii
圖三十五、鈣離子濃度對碘-125-Annexin V 及碘-125-A6L15 融合蛋
白與未稀釋的ghost 紅血球細胞在室溫下反應30 分鐘之結
合比率的影響 (n = 3) .................................................... - 89 -
圖三十六、Ghost 紅血球細胞濃度對碘-125-Annexin V 及碘
-125-A6L15 融合蛋白在鈣離子濃度8 mM,室溫下反應
30 分鐘結合比率的影響 (n = 3) ................................... - 90 -
圖三十七、Nanosep 3K OMEGA 分離管........................................ - 91 -
xiii
表目錄
表一、Annexin V 衍生蛋白的代碼及序列結構.............................. - 92 -
表二、鎝-99m-AV-47 放射化學數據匯整........................................ - 93 -
表三、鎝- 99m-AV-47R 放射化學數據匯整..................................... - 94 -
表四、鎝-99m-AV-3 放射化學數據匯整.......................................... - 95 -
表五、鎝-99m-AV-128 放射化學數據匯整...................................... - 96 -
表六、碘-125-Annexin V 放射化學數據匯整.................................. - 97 -
表七、碘-125-A6L15 融合蛋白放射化學數據匯整........................ - 98 -
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