Prevention of reperfusion liver injury by SOD-(DIVEMA) conjugate

通过二乙烯基醚与马来酸酐共聚物（DIVEMA）合成平均分子量为30000和5000，分子量分布较窄（Wm/Mn=1.4）的偶联物，建立了体外和体内延长超氧化物歧化酶（SOD）活性的方法。人血浆中SOD酶活性在139 min内下降50%，而SOD- divema在24 h内没有下降。注射到小鼠体内的SOD在6分钟内失去50%的活性，而结合物中SOD的半衰期为69分钟。偶联DIVEMA可显著降低SOD的抗原性，提高SOD的温度稳定性。同时，本课题新开发了一套活体荧光外显照明电视显微观察分析系统，以评估DIVEMA偶联对肝脏微循环再灌注损伤的预防效果。用荧光素钠和罗丹明6g染色血清和白细胞。在我们新开发的外延照像显微镜下记录门静脉脱栓后30min的肝脏微循环。门静脉痉挛持续30分钟，并在剥离前1分钟给予一种测试药物。灌注率(No。每针灌注正弦波数/正弦波总数)、小叶内黏附和滚动白细胞数、黏附和滚动白细胞率(No。黏附和滚动白细胞数/总数以肝窦后中央小静脉内通过的白细胞数量作为肝再灌注损伤的指标。与SOD相比，SOD- divema偶联物对再灌注损伤的预防作用显著（p<0.05）。另一方面，与未处理的对照相比，SOD在各施用指标上的微循环表现不显著。

Procedures for prolongation of superoxide dismutase (SOD) activity both in vitro and in vivo were established by synthesizing the conjugates with the copolymer of the divinyl ether and maleic anhydride (DIVEMA), which have average molecular weigh of 30,000 and 5,000 and narrow molecular weigh distribution (Wm/Mn=1.4). The enzymatic activity of SOD decreased to 50% within 139 min.in human plasma, on the contrary SOD-DIVEMA showed no decrease in 24 hr. SOD injected into mice lost 50% of its activity within 6 min., whereas a half life of SOD in conjugate was 69 min. The conjugating of DIVEMA markedly diminished antigenicity of SOD and increased the stability for temperature. While, an intravital fluorescence epi-illuminated television microscopic observation and analyzing system was newly developed by this Grant in order to estimate the efficacy of conjugating with DIVEMA on the prevention of reperfusion injury of the liver microcirculation. Sodium fluorescein and Rodamin-6G were used for staining serum and leukocyte. The liver microcirculation was recorded under our newly developed epi-illumination video microscope for 30min.after decramping the portal vein. Cramping of the portal vein was continued for 30 min.and a tested drug was administered one min.prior to decramping. The perfusion rate (No.of perfusing sinusoid per acine/Total number of sinusoid), the number of sticking and rolling leukocytes in the lobulus, and the rate of sticking and rolling leukocytes (No.of sticking and rolling leukocytes/total No.of passsing leukocyte) in the post sinusoidal central venule were used as indicators for the reperfusion injury of the liver. Administration of SOD-DIVEMA conjugate prevented reperfusion injury significantly (p<0.05) in compare to SOD administration. On the other hand SOD showed insignificantly better microcirculation estimated in every applied indicators than non treated control.