鎝99m(I)-三羰基標誌組胺酸耦合Annexin V作為細胞凋亡造影之研究

Abstract

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.