ABSTRACT
Objective To evaluate myocardial apoptosis with 99Tcm-C2A-GST myocardial imaging using the recombined C2A domain of Synaptotagmin Ⅰ by gene engineering. Methods ( 1 ) The C2A gene was inserted into the prokaryotic glutathione S-transferate (GST) fusion protein expression plasmid pGEX-6P-1. The recombinant plasmid was transformed into E. coli BL21. C2A-GST fusion protein was purified after BL21 was induced with isopropyl-β-D-1-thiogalactopyranoside (IPTG). (2)The activity of fusion protein was identified by cell binding test with fluorescein-5-isothiocyanate (FITC)-C2A-GST. (3) The C2A-GST fusion protein was labeled with 99Tcm using 2-iminothiophene hydrocoride method. Radiochemical purity was determined with thin layer chromatography. (4)99Tcm-C2A-GST (7.4 MBq) was injected to ischemia-reperfusion rat models through tail vein. The image was acquired with SPECT at 1 h after injection, and then hearts were removed, rinsed with saline and dyed with triphenyl tetrazolium coride (TTC). The ischemic myocardium was separated from the viable myocardium and was weighted. Its radioactivity was measured by gamma counting. The difference of uptake of radiotracer between ischemic myocardium and normal myocardium was compared using percentage activity of injected dose per gram of tissue ( % ID/g) with standard deviation. SPSS 12.0 and t-test were used for data analysis. Results ( 1 ) C2A-GST fusion protein wassuccessfully expressed and its relative molecular weight was 3.8 × 104. (2) FITC-C2A-GST binding to apoptotic cells could be observed by fluorescent microscopy. (3) The radiochemical purity of 99Tcm-C2A-GST was (98.90 ±0.43)%. (4)The imaging studies showed that there was focal uptake of radioactivity in the ischemic myocardium. In vitro uptake of 99Tcm-C2A-GST was (2.41 ±0.32) % ID/g by the ischemic myocardium, however 99Tcm-C2A-GST-N-hydroxysuccinimide (C2A-GST-NHS) was (0. 82 ± 0. 24) % ID/g. There was statistically significant difference between those two groups (t = 10. 6, P ＜0.01 ). Conclusion The C2A domain of Synaptotagmin Ⅰ expressed by gene engineering can be used as the tracer for noninvasive detection of ischemic myocardium in the ischemia-reperfusion rat model.
ABSTRACT
Objective Precondition is an approach to myocardial protection during ischemia-reper-fusion by inhibiting myocardial cell apoptosis.The purpose of this study was to evaluate the cardioprotective effect using 99Tcm-syuaptotagmin I (Syt I)-C2A to detect myocardial cell apoptosis in the myocardial is-chemia-repedusion rat model.Methods (1) The C2A domain of Syt I was labeled with 99Tcm using 2-iminothiophene hydrochloride (IT) method.Radiochemical purity was determined with thin layer chroma-tography.The binding activity of radiolabeled protein was assessed using eamptothecin-treated Jurket cells.(2) One group of 6 rats was prepared for myocardial ischemia-reperfusion model(A group),and another group of 6 rats was prepared for myocardial ischemia precondition model(B group).99Tcm-Syt I-C2A was injected via the tail vein at a dosage of about 7.4 MBq.At 1h after injection,the rat was sacrificed,and the heart was removed to rinse with saline and dye with triphenyl tetrazolium eoride (TTC).According to the resdt of myocardial dye,theischemic myoeardium was separated from the viable myocardium and weight was measured,and then its radioactivity was determined by gamma counting.The difference of radioactive uptake in the ischemic myocardium between these two group models was compared using percentage activity of injection dose per gram of tissue(%ID/g)±standard deviation(x±s).SPSS 12.0 was used for data analy-sis,and t-test was used to compare data.Results (1) The radiochemical purity of 99Tcm-Syt I-C2A was (98.90±0.43)%,and the radioactivity in the camptothecin-treated group was (10.99±0.55) folds higher than that of non-treated viable control group.(2)In the ischemia-reperfusion model,the radioactive uptake of 99Tcm-Syt,I-C2A was(2.41±0.32)%ID/g in the ischemic myocardium,and(0.16±O.02)%ID/g in the nomud myocardiunm.However,in the myocardial ischemia precondition model,(0.46±0.05)%ID/g in the isehemic myocardium was measured,and(0.20±0.05)%ID/g in the normal myocardium.Uptake of 99Tcm-Syt I-C2A in ischemic myocrdium showed statistically significant difference (t=8.52,P<O.01) between these two groups of models.Conclusion 99Tcm-C2A-glutathione S-transferase (GST) may be used to quantita-tively detect cardioprotective effect of ischemic precondition,which could inhibit myocardium apoptosis on ischemic myocardium in the ischemia-reperfusion rat model.