| Apoptosis is an intimate component of normal organ function, which keeps the whole body balance. The process of programmed cell death, or apoptosis, has been one of the most intensively studied topics in the biological sciences during the last 3 decades. One of the important characteristics of the early apoptosis is the exteriorization of phosphotidy lserine (PS). Annexin V has high affinity for PS exposed on the cell membrane, which become the important marker of detection of early apoptosis. The in vivo imaging research of radiolabeled Annexin V have made great progress in recent years, which would have been improving the comprehension about disease, prediction of response to disease therapy and development of new drugs so on.Based on the results reported in literature, the domestic recombinant human Annexin V have been radiolabeled and the feasibility of in vivo imaging apoptosis with those labeled compounds have been evaluated. The results, which have been accomplished, are list as:1. Preparation and purification of Annexin VThe technique procedure concerned in the preparation and purification of Annexin V was established: the temperature and time of vector expression, the basic routine and purification of proteins. The results of SDS-PAGE analysis showed that the mature Annexin V with high purity was obtained by ion exchange chromatography.2 Preparation of 131/125I-Annexin VThe labeled conditions and the stability of 131/125I-Annexin V had been investigated, while the analytical methods of TLC and HPLC had been established. The labeling yield of 131/125I-Annexin V was over 80% with Iodo-Gen method and up to 95% after PD-10 purification with good stability at room temperature. The radiochemical purity was determined by ITLC-SG with butyl alcohol/ethanol/ ammonia (5/1/2), Rf of 131/125I-Annexin V was 0.0-0.1. HPLC: GF250 column, 0.01 mmol/L PBS elute, flow ratel .Oml/min, retention time of 131/125I-Annexin V was about 6.3min.3 Preparation of 99mTc-HYNIC-Annexin VBesides the synthesis of Succinimidyl 6-HYNIC (S-HYNIC) and linking of S-HYN1C with Annexin V, the preparation of 99mTc-HYNIC-Annexin V had been finished, while the analytical methods of TLC and HPLC had been established. (1) S-HYNIC had been synthesized and characterized by IR, 1H-NMR and elemental analysis. The three key problems (DMF remove, anhydrous condition and BOC remove) had been solved during the synthetic process. (2) Coupling conditions: 1mol Annexin V (5.5mg/mL) was derivatized with 6mol S-HYNIC in 20mmol/L HEPES for 5hr shielded from light, one Annexin V coupled two HYNIC. The coupling conditions ofAnnexin V concentration and reacting solvent had been systematically investigated. (3) The 99mTc-HYNIC-Annexin V had been obtained via "3+1" mixed-ligand approach by using Tricine as co-ligand and SnCl2 as deoxidized reagent, its radiochemical purity was over 95%. The specific activity of 26.8 MBq/ g Annexin V was 2 times over than the reported value of literature.4. Evaluation of Biology and Animal ExperimentThe in vitro/vivo biological property and animal experiment of l31/125I-Annexin V and 99mTc-HYNIC-Annexin V had been investigated. (1) 125I-Annexin V could detect the in vitro apoptosis successfully, which was consistent with FITC- Annexin V by flow cytometry. These findings implied that 125I-Annexin V could be used for the radioimmuoassay in vitro apoptosis. (2) The biological activity of Annexin V for apoptotic cell was determined by the cell binding Assay: Ko-8.53?.30nmol/L, RT= 8.79?.21nmol/L. (3) The biodistribution of normal mice showed that 125I-Annexin V was concentrated in spleen, kidney and liver and 99mTc-HYNIC-Annexin V was concentrated in spleen, kidney, liver, and lung. The deiodination of 125I-Annexin V was found; 99raTc-HYNlC-Annexin V was excreted by kidneys. (4) Through the biodistribution and imaging of animal model (SD rats after injection of 150mg/kg cyclophosphamide), intravenously administered radiolabeled Annexin V localized preferentially in femur, vertebrae and spleen. The maximum uptake in spleen was visualized at...
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