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研究生:張淑敏
研究生(外文):Shu-Min Chang
論文名稱:三羰基鎝99m標誌HYNIC-AnnexinV耦合物作為細胞凋亡造影劑之研究
論文名稱(外文):99mTc(I)Tricarbonyl Labeled HYNIC-AnnexinV as a Potential Apoptosis Imaging Agent
指導教授:羅建苗
指導教授(外文):Jam-Mau Lo
學位類別:碩士
校院名稱:國立清華大學
系所名稱:原子科學系
學門:工程學門
學類:核子工程學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:53
中文關鍵詞:三羰基鎝99m細胞凋亡磷脂絲氨酸
外文關鍵詞:99mTc(I)Tricarbonylapoptosisphosphatidylserine
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在細胞凋亡的研究中,利用螢光劑及γ線核種標誌Annexin V可作為細胞凋亡的體外及體內偵測。99mTc 以99mTc(V)O3+型態,藉由雙官能基螯合劑hydrazinonicotinamide (HYNIC)標誌於Annexin V作為偵測細胞凋亡試劑文獻上已有諸多報導。本實驗欲利用99mTc三羰基錯合離子[99mTc(CO)3(OH2) 3] +為前驅物,取代99mTc(V)O3+,將99mTc標誌於HYNIC-Annexin V上作為細胞凋亡造影劑。由於[99mTc(CO)3(OH2)3] +容易且穩定地與兩個具供電子之氮原子之螯合劑( HYNIC)形成錯合物,藉此一新鎝99m標誌法值得發展為細胞凋亡造影劑。
參考過去文獻報導方法分別製備出Annexin V與HYNIC耦合物與[99mTc(CO)3(OH2)3]+ [Blankenberg et al. PNAS 11(1995)6349 and Alberto et al. J Am Chem Soc 120(1998) 7987]。以[99mTc(CO)3(OH2) 3] +為前驅物於pH7.4及室溫條件下進行標誌HYNIC-Annexin V,所得之99mTc(I)HYNIC- Annexin V再經離心濃縮管純化。利用放射化學分析方法包括薄層層析法、電泳動和高效能液相層析測定標誌效率與穩定度。99mTc(I)HYNIC- Annexin V對生物分子結合活性影響之測試,以人類凋亡的肝癌細胞(HepG2)為對象藉由與螢光劑標誌的Annexin V作競爭結合實驗。為了探討在活體內的生物分佈研究,以抗Fas抗體引發Balb/c小鼠的肝細胞凋亡,2小時後再注射99mTc(I)HYNIC- Annexin V。注射後分別於不同時間點將小鼠犧牲,取其組織經生理食鹽水醒秤重後以自動加馬閃爍計數器度量放射活度。
經由放射化學分析結果顯示[99mTc(CO)3(OH2) 3] +標誌HYNIC- Annexin V之放射化學純度達90%以上,1小時內在室溫下於PBS緩衝液和血清中的穩定度相當高,其放射化學純度可維持在90%,4小時後仍可達70%。正常小鼠之生物分佈實驗結果顯示肝臟有較高攝取率,30分鐘達到最大值5.31%(%I.D./g),其次是腎臟,相較之下其他的組織器官則甚低,但血液的滯留時間較長,1小時後仍有0.97%。至於對引發肝細胞凋亡之小鼠,注射後1、2及3小時其肝臟對99mTc(I)HYNIC-Annexin V的攝取率分別為10.04%、8.77%、9.35%,較正常老鼠之1、2及3小時肝臟攝取率4.67%、3.89、3.21增加約2倍,而腎臟在1小時時間點上其攝取率由3.12%減至2.02%減少30%。
由放射化學分析及活體內與體外的生物實驗結果證實,[99mTc(CO)3(OH2)3] +可有效穩定地標誌HYNIC-Annexin V,不會造成Annexin V因放射線或化學反應而降解影響其對凋亡細胞之結合。99mTc(I)HYNIC-Annexin V對肝臟凋亡細胞的特異性結合表現,對未來在凋亡細胞的造影有其潛力。
Objectives: AnnexinV labeled either with radionuclide with emission or with fluorescein has been known to be capable as the agent for detecting apoptosis..99mTc by Tc(V)O3+ labeled Annexin V via hydrazinonicotin- amide as a bifunctional chelator (99mTc(V)HYNIC-Annexin V )has been reported previously as the apoptosis imaging agent. In this study, the 99mTc(I) tricarbonyl ion, 99mTc(I) (CO)3+ ion, instead of 99mTc(V)O3+, labeled HYNIC-Annexin V is developed to image apoptosis. Since [99mTc(CO)3(OH2) 3] +is easily and kinetically fast complexed with HYNIC with 2N atom donors, the novel[99mTc(CO)3(OH2) 3+] labeling to HYNIC-Annexin V may lead to a better apoptosis imaging agent.
Methods: The conjugate of AnnexinV coupled with HYNIC and [99mTc(CO)3(OH2) 3] +were prepared respectively according to the previous reports[Blankenberg et al. PNAS 11(1995)6349 and Alberto et al. J Am Chem Soc 120(1998) 7987]. [99mTc(CO)3(OH2) 3] +labeled HYNIC- Annexin V was prepared by mixing the[99mTc(CO)3(OH2) 3] +solution at pH7.4 with HYNIC-Annexin V at 25˚C. The obtained 99mTc(I)HYNIC- Annexin V was further purified by a centrifugal concentrator. Radioanalytical methods were executed including size-exclusion HPLC, TLC and electrophoresis to characterize the 99mTc(I)HYNIC-AnnexinV prepared. Apoptotic cell binding ability was assessed by a competitive binding assay with fluorescein-Annexin V in HepG2 human hepatoma cells undergoing apoptosis in starvation. Balb/c mice were hepatocyte apoptosis induced by administrating antiFas antibody and thereafter were injected with 99mTc(I)HYNIC-Annexin V. At 10, 30, 60, 120 and 180 min postinjection, the mice were sacrificed and the major tissues and organs were excised, rinsed in saline, weighed, and transferred to an auto counter for activity measurement. Results: The prepared 99mTc(I)HYNIC-Annexin V was assayed by a size- exclusion HPLC and found to have a radiochemical purity as high as 90% . 99mTc(I)HYNIC-Annexin V was observed to be stable either in PBS or in serum within 1h but to decrease to a purity at 70% at 4h after preparation. In the biodistribution study, the major uptake and retention of 99mTc(I)HYNIC-Annexin V was found in liver and kidneys, with other organs showing very much lower level of radioactivity. The uptake of 99mTc(I)HYNIC-Annexin V in the liver of the mice bearing with hepatocyte apoptosis was apparently at least two fold higher than the case in the normal mice.
Conclusions: [99mTc(CO)3(OH2) 3] +can be efficiently and stably labeled with HYNIC-Annexin V. The effectiveness of the novel prepared 99mTc(I)-HYNIC-Annexin V for imaging apoptosis has been evidenced by in vitro and in vivo experiments in this study .
中文摘要-------------------------------------------------Ⅰ
英文摘要-------------------------------------------------Ⅲ
謝誌-----------------------------------------------------Ⅴ
目錄-----------------------------------------------------Ⅶ
表目錄---------------------------------------------------XI
圖目錄--------------------------------------------------XII
第一章 緒論----------------------------------------------1
1-1 細胞凋亡-------------------------------------1
1-1-1 細胞凋亡的生理意義----------------------2
1-1-2 細胞凋亡的機制--------------------------3
1-1-3 偵測細胞凋亡----------------------------5
1-1-3˙1 體外偵測 ---------------------------5
1-1-3˙2 體內偵測----------------------------6
1-2 99mTc(I)三羰基標誌Annexin V--------------------8
1-3 研究方向及目的-------------------------------10
第二章 有機合成-----------------------------------------12
2-1 試藥----------------------------------------12
2-2 儀器與材料----------------------------------12
2-3 S-HYNI之合成-------------------------------13
2-3-1 DCC反應原理--------------------------13
2-3-2 實驗步驟-------------------------------14
2-4 結果與討論----------------------------------15
第三章 HYNIC-annexin V之製備與分析---------------------20
3-1 試藥----------------------------------------20
3-2 儀器與材料----------------------------------20
3-3 Annexin V之處理與定量-----------------------21
3-3-1透析及濃縮------------------------------21
3-3-2 蛋白質定量-----------------------------21
3-4 S-HYNIC與蛋白質Annexin V耦合反應與分析-----22
3-4-1 反應步驟-------------------------------22
3-4-2 高效能液相層析-------------------------22
3-5 結果與討論----------------------------------23
第四章 鎝99m三羰基錯合物[99m Tc(CO)3(OH2)3] +標誌HYNIC- AnnexinV----------------------------------------26
4-1 試藥與材料----------------------------------26
4-2 儀器與器材----------------------------------26
4-3 [99m Tc(CO)3(OH2)3] +之製備與標誌HYNIC Annexin V
--------------------------------------------27
4-4 標誌效率分析--------------------------------28
4-4-1 薄層層析法------------------------------28
4-4-1.1 [99m Tc(CO)3(OH2)3] +薄層層析---------28
4-4-1. 2 Tc(I)HYNIC-Annexin V薄層層析------28
4-4-2 電泳動分析-----------------------------28
4-4-2.1 [99m Tc(CO)3(OH2)3] +電泳動分析-------28
4-4-2.2 99m Tc(I)-HYNIC-Annexin V電泳動分析-29
4-4-3 高效能液相層析-------------------------29
4-4-3˙1 [99m Tc(CO)3(OH2)3] +高效能液相層析---29
4-4-3˙2 99m Tc(I)-HYNIC-Annexin V高效能液相層析---------------------------------29
4-5 穩定度試驗----------------------------------30
4-5-1 PBS穩定度試驗-------------------------30
4-5-2 血清穩定度試驗-------------------------30
4-6 生物活性分析(IC50)---------------------------30
4-6-1 定量細胞數-----------------------------31
4-6-2 HYNIC- AnnexinV耦合物活性分析(IC50)----31
4-7 結果與討論----------------------------------32
第五章 動物組織器官之生物分佈試驗-----------------------41
5-1 藥品----------------------------------------41
5-2 儀器----------------------------------------41
5-3 實驗動物------------------------------------41
5-3-1 正常動物之生物分佈---------------------41
5-3-2 肝細胞凋亡動物之生物分布---------------42
5-4 結果與討論----------------------------------42
第六章 結論---------------------------------------------48
第七章 參考文獻-----------------------------------------49
表目錄
表1 不同細胞數與FITC-Annexin V之結合量-----------------35
表2 99m Tc(I)HYNIC-AnnexinV 與細胞之結合能力------------35
表3 99mTc(I)HYNIC-AnnexinV於正常小鼠體內之生物分佈------46
表4 99mTc(I)HYNIC-AnnexinV 於小鼠體內之生物分佈---------47
圖目錄
圖1-1 Tc(I)-HYNIC- AnnexinV之化學結構式-----------------10
圖2-1 HYNIC 之1H核磁共振光譜圖------------------------18
圖2-2 6-BOC—HYNIC之1H核磁共振光譜圖------------------18
圖2-3 Succinimidyl 6-BOC—HYNIC之1H核磁共振光譜圖-------19
圖2-4 Succinimidyl-6-HYNIC 之1H核磁共振光譜圖-----------19
圖3-1 BCA assay (Annexin V)之標準校正曲線----------------24
圖3-2 BCA assay (HYNIC-Annexin V ) 之標準校正曲線--------24
圖3-3 HYNIC-Annexin V 之(SE)HPLC分析圖譜(波長: 280nm)--25
圖4-1 [99mTc(CO)3(OH2)3] +之薄層層析圖---------------------36
圖4-2 Tc(I)HYNIC-AnnexinV之薄層層析圖------------------36
圖4-3 [99mTc(CO)3(OH2)3] +之電泳動分析圖-------------------37
圖4-4 Tc(I)HYNIC-AnnexinV之電泳動分析圖----------------37
圖4-5 [99mTc(CO)3(OH2)3] +高效能液相層析圖-----------------38
圖4-6 Tc(I)-HYNIC-Annexin V高效能液相層析----------------38
圖4-7 Tc(I)HYNIC-AnnexinV於pH7.2 血清中穩定度測試------39
圖4-8 Tc(I)HYNIC-AnnexinV於pH7.2 PBS buffer之穩定度測試-40
圖 5-1 [99mTc(CO)3(OH2)3] 與+[99mTc(CO)3(HYNIC)] +不同時間之血液活度比-----------------------------------------45
圖 5-2 99mTc(I)HYNIC-AnnexinV於正常小鼠體內之器官劑量分佈圖-----------------------------------------------46
圖 5-3 99mTc(I)HYNIC-AnnexinV 於肝凋亡細胞小鼠體內之生
物分佈-------------------------------------------47
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