細胞凋亡(apoptosis)，又稱細胞計畫性死亡(celled programmed
cell death)，無論在正常生理或病變過程中都扮演著相當重要的角
色。細胞凋亡發生初期， 原本位於細胞膜內的磷脂絲胺酸
(phosphatidylserine, PS) 受到凋亡蛋白酶 (caspase) 的調控會轉移到細胞膜外。而人體內生性的蛋白質annexin V 和磷脂絲胺酸有很高的親和力。基於此，放射性標誌annexin V 可發展為細胞凋亡的造影藥物， 在文獻上已有許多報導， 其中藉由雙官能基螯合劑
hydrazinonicotinamide (HYNIC) 標誌99mTc 於annexin V 藥物
(99mTc(V)-HYNIC-annexin V) 已經進入人體實驗。本研究參考過去文獻所報導的方法，利用分生技術合成在N端接有6 個組胺酸 (histidine)的annexin V (簡稱his-annexin V)，進而以鎝99m(I)三羰基離子標誌his-annexin V。
在實驗上，我們建立在N 端帶有6 個組氨酸 (histidine) 的
pETBlue-1/his-annexin V 質體，接著轉型至E. coli 使大量表現 hisannexin V。利用Ca2+離子做為可逆性 (reversible) 的媒介來溶解質含體(inclusion body)，可有效的得到大量的水溶性(soluble)目標蛋白質，最後再使用親和層吸管柱Ni2+ sepharose column 予以純化。由本研究結果，我們成功地獲得高產率 (39 mg/l) 高純度 (~98%) 的his-annexin V。
在鎝99m 標誌his-annexin V 之試驗，利用鎝99m(I)三羰基離子，
[99mTc(CO)3(OH2)3]+為前驅物標誌his-annexin V，藉由鎝99m(I)
三羰基離子與組胺酸配位鍵結， 可得高標誌產率之
99mTc(I)-his-annexin V，以薄層層析(TLC)、高效能液相層析 (HPLC)和分子大小排阻層析(SEC) 等測定標誌產率、放化純度和穩定度。由本實驗結果得知99mTc(I)- his-annexin V 之標誌產率可達87%以上，且在PBS 和血清中具有高度的穩定性，值得進一步發展為細胞凋亡之造影劑。

Apoptosis, also called programmed cell death, plays an important role in normal physiology and many disease processes. The early indication of apoptosis is the translocation of phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the plasma membrane. Once
exposed to the extracellular environment, PS will display available sites for binding with annexin V, which is an endogenous 35-36 kDa,Ca2+-dependent, phospholipid binding protein. Due to the high affinity for PS, annexin V has proven useful for detecting the early stage of apoptosis.
Radiolabeled annexin V has been studied extensively for the
detection of apoptosis in both animals and humans; most clinical experience has been gained with a 99mTc-labeled annexin V complex modified with a hydrazinenicotinamide ligand (99mTc(V)-HYNICannexin V).
In this study, it has been attempted to develop a form of annexin V constructed with N-terminal extention containing six histidine residues. The his-tagged annexin V (abbreviated as his-annexin V) can be directly labeled with 99mTc(I) tricarbonyl ion, [99mTc(CO)3(OH2)3]+ .In experiment, a pETBlue-1-his-annexin V plasmid has been
constructed. The recombinant his-annexin V was allowed to overexpress in E. coli by the plasmid. Calcium ion was utilized as a reversible medium to solve the inclusion body. Finally, his-annexin V was purified by a single-step affinity chromatography via Ni2+ sepharose column. The
his-annexin V product could be obtained approximately 39 mg of protein/l of culture with a high purity of ~98% as judged by SDS-PAGE.For the 99mTc labeling, 99mTc-tricarbonyl ion, [99mTc(CO)3(OH2)3]+ , was
synthesized and utilized as the precursor to conjugate directly with his-annexin V. The resultant 99mTc(I)-his-annexin V was characterized by thin layer chromatography(TLC), high performance liquid chromatography (HPLC) and size exclusion chromatography (SEC). The labeled yield for 99mTc(I)-his-annexin V was ≧87%. 99mTc(I)-his-annexin
V exhibited good stability in PBS and serum. 99mTc(I)-his-annexin V is worthy to be be further investigated as a potent apoptosis imaging agent.

1. Kerr, J. F.; Wyllie, A. H.; Currie, A. R., Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972, 26,(4), 239-57.
2. Blankenberg, F. G.; Strauss, H. W., Non-invasive diagnosis of acute heart- or lung-transplant rejection using radiolabeled annexin V. Pediatr Radiol 1999, 29, (5),
299-305.
3. Fadok, V. A.; Voelker, D. R.; Campbell, P. A.; Cohen, J. J.; Bratton, D. L.;Henson, P. M., Exposure of phosphatidylserine on the surface of apoptotic
lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1992, 148, (7), 2207-16.
4. Zwaal, R. F.; Schroit, A. J., Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997, 89, (4), 1121-32.
5. Williamson, P.; Schlegel, R. A., Back and forth: the regulation and function of transbilayer phospholipid movement in eukaryotic cells. Mol Membr Biol 1994, 11,
(4), 199-216.
6. Schroit, A. J.; Zwaal, R. F., Transbilayer movement of phospholipids in red cell and platelet membranes. Biochim Biophys Acta 1991, 1071, (3), 313-29.
7. Bevers, E. M.; Comfurius, P.; Dekkers, D. W.; Zwaal, R. F., Lipid translocation across the plasma membrane of mammalian cells. Biochim Biophys Acta 1999, 1439,(3), 317-30.
8. Martin, S. J.; Reutelingsperger, C. P.; McGahon, A. J.; Rader, J. A.; van Schie, R.C.; LaFace, D. M.; Green, D. R., Early redistribution of plasma membrane
phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 1995, 182, (5),
1545-56.
9. Van den Eijnde, S. M.; Boshart, L.; Reutelingsperger, C. P.; De Zeeuw, C. I.;Vermeij-Keers, C., Phosphatidylserine plasma membrane asymmetry in vivo: a pancellular phenomenon which alters during apoptosis. Cell Death Differ 1997, 4, (4),311-6.
10. Denecker, G.; Dooms, H.; Van Loo, G.; Vercammen, D.; Grooten, J.; Fiers, W.;Declercq, W.; Vandenabeele, P., Phosphatidyl serine exposure during apoptosis precedes release of cytochrome c and decrease in mitochondrial transmembrane potential. FEBS Lett 2000, 465, (1), 47-52.
11. Green, A. M.; Steinmetz, N. D., Monitoring apoptosis in real time. Cancer J 2002, 8, (2), 82-92.
12. Blankenberg, F.; Narula, J.; Strauss, H. W., In vivo detection of apoptotic cell death: a necessary measurement for evaluating therapy for myocarditis, ischemia, and
heart failure. J Nucl Cardiol 1999, 6, (5), 531-9.
13. Creutz, C. E.; Pazoles, C. J.; Pollard, H. B., Identification and purification of an adrenal medullary protein (synexin) that causes calcium-dependent aggregation of isolated chromaffin granules. J Biol Chem 1978, 253, (8), 2858-66.
14. Gerke, V.; Moss, S. E., Annexins: from structure to function. Physiol Rev 2002,82, (2), 331-71.
15. Cookson, B. T.; Engelhardt, S.; Smith, C.; Bamford, H. A.; Prochazka, M.; Tait, J.F., Organization of the human annexin V (ANX5) gene. Genomics 1994, 20, (3),463-7.
16. Huber, R.; Romisch, J.; Paques, E. P., The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.Embo J 1990, 9, (12), 3867-74.
17. Bohn, H.; Kraus, W., [Isolation and characterization of a new placenta specific protein (PP10) (author's transl)]. Arch Gynecol 1979, 227, (2), 125-34.
18. Reutelingsperger, C. P.; Hornstra, G.; Hemker, H. C., Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord. Eur J Biochem 1985, 151, (3), 625-9.
19. Swairjo, M. A.; Seaton, B. A., Annexin structure and membrane interactions: a molecular perspective. Annu Rev Biophys Biomol Struct 1994, 23, 193-213.
20. Weinman, S., Calcium-binding proteins: an overview. J Biol Buccale 1991, 19,(1), 90-8.
21. van Heerde, W. L.; de Groot, P. G.; Reutelingsperger, C. P., The complexity of the phospholipid binding protein Annexin V. Thromb Haemost 1995, 73, (2), 172-9.
22. Andree, H. A.; Reutelingsperger, C. P.; Hauptmann, R.; Hemker, H. C.; Hermens,W. T.; Willems, G. M., Binding of vascular anticoagulant alpha (VAC alpha) to planar
phospholipid bilayers. J Biol Chem 1990, 265, (9), 4923-8.
23. Tzima, E.; Walker, J. H., Platelet annexin V: the ins and outs. Platelets 2000, 11,(5), 245-51.
24. Benz, J.; Hofmann, A., Annexins: from structure to function. Biol Chem 1997,378, (3-4), 177-83.
25. Delmer, D. P.; Potikha, T. S., Structures and functions of annexins in plants. Cell Mol Life Sci 1997, 53, (6), 546-53.
26. Romisch, J.; Schuler, E.; Bastian, B.; Burger, T.; Dunkel, F. G.; Schwinn, A.;Hartmann, A. A.; Paques, E. P., Annexins I to VI: quantitative determination in
different human cell types and in plasma after myocardial infarction. Blood Coagul Fibrinolysis 1992, 3, (1), 11-7.
27. Walker, J. H.; Boustead, C. M.; Koster, J. J.; Bewley, M.; Waller, D. A., Annexin V, a calcium-dependent phospholipid-binding protein. Biochem Soc Trans 1992, 20,
(4), 828-33.
28. Kirsch, T.; Harrison, G.; Golub, E. E.; Nah, H. D., The roles of annexins and types II and X collagen in matrix vesicle-mediated mineralization of growth plate cartilage. J Biol Chem 2000, 275, (45), 35577-83.
29. Matteo, R. G.; Moravec, C. S., Immunolocalization of annexins IV, V and VI in the failing and non-failing human heart. Cardiovasc Res 2000, 45, (4), 961-70.
30. Walker, J. H.; Boustead, C. M.; Brown, R.; Koster, J. J.; Middleton, C. A., Tissue and subcellular distribution of endonexin, a calcium-dependent phospholipid-binding
protein. Biochem Soc Trans 1990, 18, (6), 1235-6.
31. Tait, J. F.; Gibson, D.; Fujikawa, K., Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family. J Biol Chem 1989, 264, (14), 7944-9.
32. Zhang, G.; Gurtu, V.; Kain, S. R.; Yan, G., Early detection of apoptosis using a fluorescent conjugate of annexin V. Biotechniques 1997, 23, (3), 525-31.
33. Kemerink, G. J.; Liu, X.; Kieffer, D.; Ceyssens, S.; Mortelmans, L.; Verbruggen,A. M.; Steinmetz, N. D.; Vanderheyden, J. L.; Green, A. M.; Verbeke, K., Safety,
biodistribution, and dosimetry of 99mTc-HYNIC-annexin V, a novel human recombinant annexin V for human application. J Nucl Med 2003, 44, (6), 947-52.
34. Lahorte, C.; Slegers, G.; Philippe, J.; Van de Wiele, C.; Dierckx, R. A., Synthesis and in vitro evaluation of 123I-labelled human recombinant annexin V. Biomol Eng
2001, 17, (2), 51-3.
35. Ke, S.; Wen, X.; Wu, Q. P.; Wallace, S.; Charnsangavej, C.; Stachowiak, A. M.;Stephens, C. L.; Abbruzzese, J. L.; Podoloff, D. A.; Li, C., Imaging taxane-induced tumor apoptosis using PEGylated, 111In-labeled annexin V. J Nucl Med 2004, 45, (1),108-15.
36. Ito M, T. K., Takahashi N,, Development of a new ligand, 11C-labeled annexin V,for PET imaging of apoptosis [abstract]. J Nucl Med 2002, 43, (5), 362P,Development of a new ligand, 11C-labeled annexin V, for PET imaging of apoptosis[abstract].
37. Zijlstra, S.; Gunawan, J.; Burchert, W., Synthesis and evaluation of a 18F-labelled recombinant annexin-V derivative, for identification and quantification of apoptotic cells with PET. Appl Radiat Isot 2003, 58, (2), 201-7.
38. McQuade P, J. L., Vanderheyden JL, Welch MJ, 94mTc and 64Cu labeled annexin-V, positron emitting radiopharmaceuticals to study apoptosis [abstract]. J
Nucl Med 2003, 46, (Supppl 1), S335.
39. Smith-Jones PM, A. A., Zanzonico P, Tait J, Larson SM, Strauss HW, 68Ga labelling of annexin-V: comparison to 99mTc-annexin-V and 67Ga-annexin [abstract]. J Nucl Med 2003, 44, (5), 49P-50P.
40. Keen, H. G.; Dekker, B. A.; Disley, L.; Hastings, D.; Lyons, S.; Reader, A. J.;Ottewell, P.; Watson, A.; Zweit, J., Imaging apoptosis in vivo using 124I-annexin V and
PET. Nucl Med Biol 2005, 32, (4), 395-402.
41. Tait, J. F.; Brown, D. S.; Gibson, D. F.; Blankenberg, F. G.; Strauss, H. W.,Development and characterization of annexin V mutants with endogenous chelation sites for (99m)Tc. Bioconjug Chem 2000, 11, (6), 918-25.
42. Mukherjee, A.; Kothari, K.; Toth, G.; Szemenyei, E.; Sarma, H. D.; Kornyei, J.;Venkatesh, M., 99mTc-labeled annexin V fragments: a potential SPECT radiopharmaceutical for imaging cell death. Nucl Med Biol 2006, 33, (5), 635-43.
43. Cauchon, N.; Langlois, R.; Rousseau, J. A.; Tessier, G.; Cadorette, J.; Lecomte,R.; Hunting, D. J.; Pavan, R. A.; Zeisler, S. K.; van Lier, J. E., PET imaging of
apoptosis with (64)Cu-labeled streptavidin following pretargeting of phosphatidylserine with biotinylated annexin-V. Eur J Nucl Med Mol Imaging 2007,34, (2), 247-58.
44. Kasina, S.; Rao, T. N.; Srinivasan, A.; Sanderson, J. A.; Fitzner, J. N.; Reno, J.M.; Beaumier, P. L.; Fritzberg, A. R., Development and biologic evaluation of a kit for
preformed chelate technetium-99m radiolabeling of an antibody Fab fragment using a diamide dimercaptide chelating agent. J Nucl Med 1991, 32, (7), 1445-51.
45. Fritzberg, A. R.; Abrams, P. G.; Beaumier, P. L.; Kasina, S.; Morgan, A. C.; Rao,T. N.; Reno, J. M.; Sanderson, J. A.; Srinivasan, A.; Wilbur, D. S.; et al., Specific and stable labeling of antibodies with technetium-99m with a diamide dithiolate chelating agent. Proc Natl Acad Sci U S A 1988, 85, (11), 4025-9.
46. Kemerink, G. J.; Boersma, H. H.; Thimister, P. W.; Hofstra, L.; Liem, I. H.;Pakbiers, M. T.; Janssen, D.; Reutelingsperger, C. P.; Heidendal, G. A., Biodistribution
and dosimetry of 99mTc-BTAP-annexin-V in humans. Eur J Nucl Med 2001, 28, (9),1373-8.
47. Blankenberg, F. G.; Katsikis, P. D.; Tait, J. F.; Davis, R. E.; Naumovski, L.;Ohtsuki, K.; Kopiwoda, S.; Abrams, M. J.; Darkes, M.; Robbins, R. C.; Maecker, H.
T.; Strauss, H. W., In vivo detection and imaging of phosphatidylserine expression during programmed cell death. Proc Natl Acad Sci U S A 1998, 95, (11), 6349-54.
48. Roger Alberto, R. S., Andre´ Egli, and August P. Schubiger, A Novel Organometallic Aqua Complex of Technetium for the Labeling of Biomolecules:
Synthesis of [99mTc(OH2)3(CO)3]+ from [99mTcO4]- in Aqueous Solution and Its Reaction with a Bifunctional Ligand. J Am Chem Soc 1998, 120, 7987-7988.49. Egli, A.; Alberto, R.; Tannahill, L.; Schibli, R.; Abram, U.; Schaffland, A.;
Waibel, R.; Tourwe, D.; Jeannin, L.; Iterbeke, K.; Schubiger, P. A., Organometallic99mTc-aquaion labels peptide to an unprecedented high specific activity. J Nucl Med 1999, 40, (11), 1913-7.
50. Waibel, R.; Alberto, R.; Willuda, J.; Finnern, R.; Schibli, R.; Stichelberger, A.;Egli, A.; Abram, U.; Mach, J. P.; Pluckthun, A.; Schubiger, P. A., Stable one-step
technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex. Nat Biotechnol 1999, 17, (9), 897-901.51. Du, J.; Hiltunen, J.; Marquez, M.; Nilsson, S.; Holmberg, A. R., Technetium-99m
labelling of glycosylated somatostatin-14. Appl Radiat Isot 2001, 55, (2), 181-7.
52. Elegbede, A. I.; Srivastava, D. K.; Hinderliter, A., Purification of recombinant annexins without the use of phospholipids. Protein Expr Purif 2006, 50, (2), 157-62.
53. Romisch, J.; Heimburger, N., Purification and characterization of six annexins from human placenta. Biol Chem Hoppe Seyler 1990, 371, (5), 383-8.
54. Tzima, E.; Trotter, P. J.; Orchard, M. A.; Walker, J. H., Annexin V relocates to the platelet cytoskeleton upon activation and binds to a specific isoform of actin. Eur
J Biochem 2000, 267, (15), 4720-30.
55. Tait, J. F.; Smith, C.; Gibson, D. F., Development of annexin V mutants suitable for labeling with Tc(I)-carbonyl complex. Bioconjug Chem 2002, 13, (5), 1119-23.
56. Wang, F.; He, X. W.; Yan, H. L.; Huang, J. J.; Zhang, Y.; Jiang, L.; Gao, Y. J.;Sun, S. H., Non-fusion expression in Escherichia coli: Single-step purification of
recombinant human annexin A5 for detection of apoptosis. Protein Expr Purif 2006,45, (1), 80-7.