Experimental Study On Apoptosis Imaging Agent ~ (99m) Tc-annexin ¢õ

Apoptosis (PCD, program cell death) is an active death modulated bymultiple genes. It distinguishs from cell necrosis and lies in manyphysiological phenomena and pathological conditions. Recently,radiolabeled Annexin V,as a nuclear probe, often be used in detectingapoptosis in vivo.Annexin V is a member of the calcium and phospholipid bindingsuperfamily of Annexin proteins. The mature Annexin V molecule consistsof 319 amino acids with a total molecular weight of 35.8 kDa. At an earlystage of apoptosis, phosphatidylserine (PS), a constitutive membraneaminophospholipid normally presents on the membrane' s inner surface, isexposed on the outer surface. Annexin V has high affinity for chargedphospholipids inthe presence of physiological concentrations of calcium.It is a non-invasive, direct, real-time method for apoptosis monitoringof the the organism through detecting the radiolabeled Annexin V.We focus on finding a easy and high-efficiency method to manufacturemutant human annexin V with endogenous metal-chelating sites, in virtueof molecular biology.Methods:1. The expression and purification of the human annexin V in Ecoli.2. The expression and purification of the mutant human annexin V inPichia pastoris.3. The binding activity of mutant annexin V was determined withphosphatidylserine-exposed-erythrocytes and fluoresceinisothiocyanate labeled Annexin V.4. The radiolabeled method with technetium99m, and its radiolabelingyield, radiochemical purity, binding activity in vitro.5. The stability of ^9mTc-Annexin V. 6. The biodistribution of 99mTc-Annexin V in normal mice andSarcoma-grafted Mice.Results:1. Mutant human annexin V with endogenous metal-chelating sites canbe secreted by Pichia pastoris, Pichia pastoris secret annexin V into theculture supernatant, its purification can reach 95ï¼…by several simplypurificatory method, it spends little time and cost on the production ofannexin V.2. The 50ï¼…inhibitory concentration is 4nmol/l.3. The radiolabeling yield of ^99mTc-Annexin V is 60ï¼….Itsradiochemical purity can reach 92ï¼…after purification by SephadexG-25, while,its binding activity in vitro is 67ï¼….4. ^99mTc-Annexin V has a good stability. There is no significant changeon its radiochemical purity and binding activity in vitro.5. Normal mice biodistribution results indicated that ^99mTc-Annexin Vwas accumulated mainly in the kidney, liver and spleen after intravascularadministration and evacuated by kidney. Sarcoma-grafted Micebiodistribution results indicate that T/ M and T/ B increase gradually,T/ M reach 5.71 in 6.0h.Conclusion:Our lab produce human annexin V via Pichia pastoris system, thissystem have a lot of advantage than Ecoli system, such as good efficiency.Mutant annexin V avoid the molecular structure change with the absenceof Hydrazinonicotinamide. lt also provides an important basis for furtherstudy of ^99mTc-Annexin V, however, the more detailed evaluation isrequired.