神经突触结合蛋白Ⅰ-C2A区重组表达及其在心脏缺血-再灌注大鼠模型中的显像研究

Expression of C2A domain of synaptotagmin Ⅰ fusion protein and its imaging in the ischemia-reperfusion rat model

目的 利用基因工程方法重组表达神经突触结合蛋白Ⅰ的C2A片段,探讨其在心肌细胞凋亡显像中的应用价值.方法 （1）将C2A基因连接到含有谷胱甘肽转移酶（GST）的重组原核表达载体pGEX-6P-1上,转化感受态细菌BL21,异内基硫代半乳糖苷（IPTG）诱导后纯化.（2）用异硫氰酸荧光素（FITC）标记纯化的蛋白,通过细胞结合实验鉴定蛋白质活性.（3）采用2-亚氨基噻吩方法,进行99TcmO-4标记C2A-GST融合蛋白,标记后的蛋白质用纸层析法测定其放化纯.（4）制备大鼠心肌缺血-再灌注模型,通过尾静脉注射99Tcm-C2A-GST,1 h后用SPECT仪进行显像 显像结束后,处死大鼠,取出心肌,用氯化三苯基四氮唑（TTC）染色,分离缺血心肌和存活心肌,测质量及其放射性计数,比较缺血心肌与正常心肌每克组织百分注射剂量率（%ID/g）值之间差别.采用SPSS12.0软件行统计分析,数据间比较采用t检验.结果 （1）成功表达的C2A-GST蛋白,相对分子质量约为3.8×104.（2）荧光显微镜下观察FITC-C2A-GST具有结合凋亡细胞的功能.（3）标记后的99Tcm-C2A-GST放化纯为（98.90±0.43）%.（4）大鼠显像示缺血损伤心肌显影清晰,体外测定缺血心肌摄取99Tcm-C2A-GST为（2.41±0.32）%ID/g,对照组99Tcm-C2A-GST-N-羟基琥珀酰亚胺（C2A-GST-NHS）的摄取为（0.82±0.24）%ID/g,两者之间差别具有统计学意义（t=10.6,P＜0.01）.结论 通过基因工程方法重组神经突触膜蛋白Ⅰ的C2A区域,重组后其具有监测缺血-再灌注大鼠模型中的心肌凋亡的作用.

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.