臺灣博碩士論文加值系統

細胞凋亡 (apoptosis) 為細胞的程序死亡，是生物體成長、分化、免疫及體內平衡不可或缺的角色。許多疾病初期都會出現細胞凋亡的現象，因此藉由偵測細胞凋亡，可以提早發現病癥及確認治療效果。Phosphatidylserine (PS)在細胞膜外表現為細胞凋亡的一種特徵。藉由Annexin V對於PS的高度親和力，可以用來偵測細胞凋亡。螢光標幟Annexin V被發展用於細胞凋亡的體外偵測，而放射性同位素標幟Annexin V則適用於核子醫學的體內檢查。為發展鎝-99m標幟Annexin V，本研究探討：(1) HYNIC與Annexin V之耦合反應 (conjugation reaction) 及利用透析法純化HYNIC-Annexin V之合適條件；(2) 探討製備用於標幟鎝-99m-HYNIC-Annexin V之合適配方，包括不同的緩衝鹽類、配方pH值、標幟體積對標幟效率的影響及穩定性分析；(3) 探討鎝-99m-HYNIC-Annexin V與PS結合之生物特性分析。本研究以磷酸鈉鹽緩衝溶液取代文獻使用的檸檬酸鈉緩衝溶液進行透析純化HYNIC-Annexin V，改善檸檬酸鈉緩衝溶液透析產生的蛋白質沉澱問題，並將透析純化HYNIC-Annexin V的回收率由31%提高到62%。本研究發現含磷酸鈉鹽之標幟用凍晶小瓶中性配方，不論是否含檸檬酸鈉，其標幟效率明顯優於含檸檬酸鈉之酸性配方。以鎝-99m-過鎝酸鈉標幟凍晶小瓶的最適配方含HYNIC-Annexin V 100 μg、二結晶水氯化亞錫32 μg、tricine 716 μg、磷酸氫二鈉 0.479 mg、磷酸二氫鈉 0.431 mg、氯化鈉 6.995 mg，pH 7.3；最適當之標幟體積為0.5 ml，使用放射活度15 mCi標幟效率可達90%，室溫經時安定性可以維持至少6小時以上。本研究使用能表現膜外phosphatidylserine (PS) 之E.coli/EH150菌株證明製備之鎝-99m-HYNIC-Annexin V能與PS結合，而且這種結合會受到外加Annexin V蛋白質的競爭性抑制。本研究找出最適合進行鎝-99m-HYNIC-Annexin V生物活性研究的E.coli/EH150細胞數目為1×109。以size-exclusion高效率液相色層分析法分析所製備鎝-99m-HYNIC-Annexin V之放射化學純度為60%，主要的放射性不純物可能來自未完全移除之HYNIC標幟物，進一步改進HYNIC-Annexin V之純化步驟有其必要。

Apoptosis, also known as programmed cell death, is an dispensable component of living body growth and development, immunoregulation, homeostasis. The early stage of many disease has appeared apoptosis symptom; therefore, we can detect apoptosis to diagnose disease and confirm if therapeutic is success or failure. The distinct feature of apoptosis is that phosphatidylserine (PS) expressed on the outer leaflet of cell membrane, so the apoptosis is detected by specific binding of Annexin V to PS. Annexin V labeled with fluorescein dye, has been used to detect apoptosis in vitro, while the annexin V label with radioisotope is appropriate for in vivo diagnosis in nuclear medicine. For developing annexin V labeled with 99mTc, this study evaluates (1) the conjugation reaction of Annexin V with HYNIC, and the optimization dialysis to purify HYNIC-Annexin V; (2) formulation optimization of HYNIC-Annexin V kit for labeling with 99mTc, including: the buffer effect、the pH effect、the volume effect and the stability for analysis for 99mTc-HYNIC-Annexin V; (3) bioactivity assessment of 99mTc-HYNIC-Annexin V Using PS expressed E. coli/EH150 strain. This study revealed that the phosphate buffer was superior to the sodium citrate in improving the yield of HYNIC-Annexin V from 31% to 62% preventing protein from precipitation during dialysis procedure, The 99mTc labeling efficiency of neutral formulation buffered with phosphate salts was much better than that of acidic composition buffered with sodium citrate (60% vs 8%). An optimal lyophilized kit formulation of HYNIC-Annexin V was explored in this study, each kit contains HYNIC-Annexin V 100 μg、SnCl2．2H2O、tricine 716 μg、Na2HPO4 0.479 mg、NaH2PO4 0.431 mg、NaCl 6.995 mg, pH 7.3; with the optimal volume (0.5 ml) and the radioactivity up to 15 mCi, the labeling efficiency was greater than 90%, which can be lasted for at least 6hr after preparation. The strain of E. coli/EH150 which expresses PS on the outer leaflet of cell membrane was explored to confirm the characteristic binding of 99mTc-HYNIC-Annexin V to PS, which was competed with native Annexin V protein. The optimal quantity of E. coli/EH150 for 99mTc-HYNIC-Annexin V bioactivity assessment was 1 109 cells. By using size-exclusion HPLC to analyze species based on molecular size, we found that the radiochemical purity of 99mTc-HYNIC-Annexin V was about 60%, the most part of impurity maybe come from the complex of HYNIC due to incomplete removal of unconjugated HYNIC molecules, it is necessary to improve to purify HYNIC-Annexin V.

中文摘要………………………………………………………………………………I
英文摘要…………………………………………………………………………….III
圖目錄………………………………………………………………………………..V
表目錄……………………………………………………………………………...VII
第一章 序言及文獻探討
1-1 何謂細胞凋亡……………………………………………………………..1
1-1-1 細胞凋亡的定義……………………………………………………..1
1-1-2 細胞凋亡的功用……………………………………………………..2
1-1-3 細胞凋亡的機制……………………………………………………..2
1-2 Annexin V蛋白質與細胞凋亡的關係…………………………………..4
1-3 體外的方法偵測細胞凋亡……………………………………………….5
1-3-1 TUNEL……………………………………………………………....5
1-3-2 活化caspase 酵素分析……………………………………………..5
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…………………………………………….....8
1-4-1 放射性同位素標幟Annexin V……………………………………..8
1-4-2 選擇Annexin V的螯合劑………………………………………….8
1-4-3 選擇鎝-99m標幟HYNIC-Annexin V的輔助配位子…………....9
1-5 鎝-99m-HYNIC-Annexin V的應用…………………………………...10
1-6 研究目的………………………………………………………………...12
第二章 材料與方法
2-1 材料……………………………………………………………………...13
2-2 儀器及設備……………………………………………………………...14
2-3 Annexin V分析…………………………………………………………15
2-3-1 電泳分析……………………………………………………………15
2-3-2 蛋白質全光譜掃描分析……………………………………………16
2-3-3 Size-Exclusion HPLC分析………………………………………..17
2-3-4 Coomassie blue法定量Annexin V……………………………….17
2-4 HYNIC-Annexin V製備……………………………………………….18
2-4-1 S-HYNIC與Annexin V耦合反應 (Conjugation Reaction)……18
2-4-2 測量Annexin V耦合HYNIC數………………………………….19
2-4-3 HYNIC-Annexin V蛋白質全光譜掃描分析……………………...20
2-5 製備HYNIC-Annexin V凍晶小瓶……………………………………..21
2-6 鎝-99m-HYNIC-Annexin V之製備與放射化學分析…………………22
2-6-1 鎝-99m-HYNIC-Annexin V之製備……………………………….22
2-6-2 放射薄層分析法……………………………………………………22
2-6-3 Size-Exclusion HPLC分析………………………………………..22
2-7 鎝-99m-HYNIC-Annexin V的生物活性分析………………………...23
2-7-1 評估E. coli/EH150之最適當細胞數目…………………………..23
2-7-2 鎝-99m-HYNIC-Annexin 的生物活性分析……………………..24
第三章 結果
3-1 HYNIC-Annexin V製備………………………………………………25
3-2 製備鎝-99m-HYNIC-Annexin V與標幟效率分析…………………..27
3-3 鎝-99m-HYNIC-Annexin V的生物活性分析………………………..30
第四章 討論……………………………………………………………………...32
第五章 結論……………………………………………………………………...38
參考文獻…………………………………………………………………………...39

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