【作者】 杨伟；

【导师】 罗文鸿；

【作者基本信息】 汕头大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 氨基脲敏感型胺氧化酶(Semicarbazide-sensitive amine oxidase, SSAO; EC1.4.3.6)是一组含铜胺氧化酶的统称,广泛分布于器官和组织中,尤其在脂肪细胞、血管内皮细胞和平滑肌细胞中酶活性较高,它的可溶性形式也存在于血浆中。脂肪细胞和平滑肌细胞表达的SSAO酶可促进葡萄糖转运,具有类胰岛素效应;而内皮细胞表达的SSAO酶即血管黏附蛋白-1(VAP-1)参与炎症过程中白细胞的运输与外渗。生化方面,SSAO催化伯胺的氧化脱胺生成相应的醛、氨和过氧化氢(RCH2NH2 + O2 + H2O→RCHO + NH3 + H2O2)。有研究表明在离体肺缺血再灌注损伤模型中SSAO参与过多活性氧族的生成。然而SSAO在心肌缺血再灌注损伤中是否起作用尚未见报道。因此,我们用大鼠缺血再灌注模型来验证这一假设。所有SD大鼠心脏都给予缺血30 min再灌注180 min的处理。所有大鼠随机分成八组,每组十只:1)缺血再灌注对照组:缺血前后不给予任何干预;2)氨基脲处理组:实验前10 min给予氨基脲(30 mg/kg,溶解于1 ml生理盐水)处理;3)缺血预适应组:长时间缺血前给予5 min缺血10 min再灌注;4)缺血预适应+氨基脲处理组:实验前10 min给予氨基脲处理,然后长时间缺血前给予5 min缺血10 min再灌注;5)缺血后适应组:长时间再灌注前给予3个循环10 s再灌注和10 s再缺血的后处理。6)缺血后适应+氨基脲处理组:实验前10 min给予氨基脲处理,然后长时间再灌注前给予3个循环10 s再灌注和10 s再缺血的后处理。7)假手术组:实验过程同其他组,但穿线不结扎。8)假手术+氨基脲处理组:实验前10 min给予氨基脲处理,然后穿线不结扎。在本实验中,采用NBT(氯化硝基四氮唑兰)染色法测定心肌梗塞范围;比色法测定大鼠血浆肌酸激酶(CK)、丙二醛(MDA)、髓过氧化物酶(MPO)、羟自由基(·OH)等水平;高效液相色谱法测定血浆SSAO酶活性;HE染色法进行组织学评估。在本实验中我们得到如下结果:(1)各组的心肌梗塞范围为:缺血再灌注对照组(43.2±1.0%);氨基脲处理组(27.7±1.2%);缺血预适应组(29.3±2.2%);缺血预适应+氨基脲处理组(13.9±3.2%);缺血后适应组(33.6±1.9%);缺血后适应+氨基脲处理组(41.1±4.2%)。与缺血再灌注对照组相比,除了缺血后适应+氨基脲处理组没有统计学差异外,其他组均有统计学差异。(2)各组血浆肌酸激酶活力(U/ml)为:缺血再灌注对照组(2.83±0.33);氨基脲处理组(2.64±0.26);缺血预适应组(2.22±0.25);缺血预适应+氨基脲处理组(2.51±0.32);缺血后适应组(2.28±0.40);缺血后适应+氨基脲处理组(2.61±0.26);假手术组(2.26±0.40);假手术+氨基脲处理组(2.28±0.28)。各组之间没有统计学差异。(3)各组血浆丙二醛水平(nmol/ml)为:缺血再灌注对照组(2.58±0.14);氨基脲处理组(1.74±0.16);缺血预适应组(2.13±0.15);缺血预适应+氨基脲处理组(1.78±0.06);缺血后适应组(2.47±0.21);缺血后适应+氨基脲处理组(1.77±0.17);假手术组(2.11±0.12);假手术+氨基脲处理组(1.66±0.10)。与缺血再灌注对照组相比,所有氨基脲处理组血浆丙二醛水平都显著降低了;与缺血后适应组相比,所有氨基脲处理组血浆丙二醛水平也显著降低。(4)各组血浆髓过氧化物酶活性(U/L)为:缺血再灌注对照组(27.53±2.93);氨基脲处理组(14.16±1.78);缺血预适应组(14.16±1.85);缺血预适应+氨基脲处理组(13.37±1.49);缺血后适应组(13.57±2.56);缺血后适应+氨基脲处理组(12.39±1.63);假手术组(13.18±2.23);假手术+氨基脲处理组(17.90±2.61)。与缺血再灌注对照组相比,其他组血浆髓过氧化物酶活性都显著降低。(5)各组血浆羟自由基水平(U/ml)为:缺血再灌注对照组(628.4±15.7);氨基脲处理组(502.6±27.8);缺血预适应组(509.6±29.9);缺血预适应+氨基脲处理组(505.4±26.9);缺血后适应组(526.6±16.8);缺血后适应+氨基脲处理组(496.9±32.6);假手术组(502.8±25.7);假手术+氨基脲处理组(525.7±29.8)。与缺血再灌注对照组相比,其他组血浆羟自由基水平都显著降低。(6)HE染色结果表明抑制SSAO酶活性显著减少了白细胞的聚集。总之,我们的研究表明抑制SSAO酶可以缓解心肌缺血再灌注损伤。更多还原

【Abstract】 Semicarbazide-sensitive amine oxidase (SSAO, EC1.4.3.6) is a common name for a group of copper-containing amine oxidases widely distributed in organs and tissues, especially in adipocytes, endothelial and smooth muscle cells. Soluble form of SSAO is also present in blood plasma. In adipocytes and smooth muscle cells, SSAO activity stimulates glucose transport, mimicking the insulin effect, while in endothelial cells, where it is identical to vascular adhesion protein-1 (VAP-1), it is involved in leukocyte trafficking and extravasation during inflammation. Biochemically, SSAO catalyzes the oxidative deamination of primary amines to produce aldehydes, ammonia, and hydrogen peroxide (RCH2NH2 + O2 + H2O→RCHO + NH3 + H2O2). Previous studies have shown that SSAO contributes to the excessive ROS formation in the isolated rat lung with ischemia-reperfusion injury. However, it remains unclear whether inhibition of SSAO plays a role in the regulation of myocardial ischemia-reperfusion injury. Therefore, we used an open-chest rat model of left coronary artery occlusion and reperfusion to test the hypothesis whether inhibition of SSAO would attenuate reperfusion-induced myocardial injury.In anesthetized open-chest Sprague-Dawley rats, the left coronary artery (LCA) underwent 30 min ischemia and 180 min reperfusion (R). All rats were randomly divided into eight groups, each group consists of 10 animals:(1) Control: There was no intervention either before or after the prolonged occlusion;(2) Semicarbazide (Sem): Sem (30 mg/kg, dissolved in saline as a volume of 1 ml/kg), a selective SSAO inhibitor, was given 10 min prior to the experiment;(3) Ischemia preconditioning (pre-con): 5 min ischemia followed by 10 min R before the prolonged occlusion;(4) Pre-con + Sem: Sem was given 10 min prior to the experiment, 5 min ischemia then 10 min R before the prolonged occlusion;(5) Ischemia postconditioning (post-con): 3 cycles of 10 s R and 10 s re-ischemia prior to 180 min R;(6) Post-con + Sem: Sem was given 10 min prior to the experiment, and 3 cycles of 10 s R and 10 s re-ischemia prior to 180 min R;(7) Sham: the surgical procedure was identical to other groups, but the LCA ligature was not ligated;(8) Sham + Sem: Sem was given 10 min prior to the experiment, and the LCA ligature was not ligated.In this experiment, the NBT staining was used to determine myocardial infarct size; colorimetric method was used to determine plasma creatine kinase (CK) activity, plasma malondialdehyde (MDA) levels, plasma myeloperoxidase (MPO) activity, and plasma hydroxyl radicals levels; plasma SSAO activity was measured by high performance liquid chromatography (HPLC); the HE staining was used to make a histological evalution.Infarct sizes in Sem-treated group (27.7±1.2%), pre-con group (29.3±2.2%), pre-con + Sem group (13.9±3.2%) and post-con group (33.6±1.9%) were remarkably smaller than control group (43.2±1.0%). Post-con + Sem group (41.1±4.2%) showed no difference with control.Plasma CK activity (U/ml) in different groups were 2.83±0.33 (control group), 2.64±0.26 (Sem-treated group), 2.22±0.25 (pre-con group), 2.51±0.32 (pre-con + Sem group), 2.28±0.40 (post-con group), 2.61±0.26 (post-con + Sem group), 2.26±0.40 (sham group), 2.28±0.28 (sham + Sem group). There were no significant differences among all the groups.Plasma MDA levels (nmol/ml) were significantly reduced in the Sem-treated group (1.74±0.16), pre-con + Sem group (1.78±0.06), post-con + Sem group (1.77±0.17), sham + sem group (1.66±0.10) compared with control group (2.58±0.14) or post-con group(2.47±0.21). Plasma MPO activity (U/L) was found to be significantly decreased in the sem-treated group (14.16±1.78), pre-con group (14.16±1.85), pre-con + sem group (13.37±1.49), post-con group (13.57±2.56), post-con + sem group (12.39±1.63), sham group (13.18±2.23) and sham + sem group (17.90±2.61) compared with control group (27.53±2.93).Plasma hydroxyl radicals levels (U/ml) were significantly less in the sem-treated group (502.6±27.8), pre-con group (509.6±29.9), pre-con + sem group (505.4±26.9), post-con group (526.6±16.8), post-con + sem group (496.9±32.6), sham group (502.8±25.7) and sham + sem group (525.7±29.8) compared with control group (628.4±15.7).The results of HE staining showed that inhibition of SSAO significantly attenuated leukocyte infiltration.In conclusion, this study demonstrated that inhibition of SSAO protects the ischemic heart against myocardial ischemia-reperfusion injury.更多还原