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目的探讨实验性自身免疫性心肌炎(EAM)小鼠心肌内12-脂加氧酶(12-LO)的表达,外周血NO水平及其与细胞凋亡的关系。方法 4~5周龄雄性BALB/C小鼠随机分成免疫性心肌炎组(AM组)和正常对照组(C组)各24只。AM组在实验的第1、7天在腹股沟皮下注射含猪心肌球蛋白100μg的混合液0.2 ml,C组于同时间同部位注射等量混合液(不含猪心肌球蛋白)。实验的第14、21、30天随机抽取各组小鼠8只取血后处死,留取心脏标本。RT-PCR法测定小鼠心肌细胞12-LO mRNA、i NOS mRNA,硝酸还原酶法检测小鼠血清NO水平,TUNEL法检测细胞凋亡指数(AI)。结果 AM组12-LO mRNA、i NOS mRNA、NO及AI均在第21天(分别为1.53±0.27;92.74±3.99;76.44±3.01;24.97±1.54)明显高于第14天(分别为0.68±0.28;82.99±2.64;57.51±2.17;13.20±2.54)和第30天(分别为1.05±0.10;87.40±3.32;64.77±3.92;14.95±2.15),P均<0.05;与C组相比,各时间点上述指标均明显升高(均P<0.05);AM组12-LO mRNA与NO、AI呈显著正相关(r=0.882;0.877;P<0.05)。结论自身免疫性心肌炎小鼠心肌细胞12-LO表达增加,促进心肌细胞凋亡及病情进展;12-LO可能通过氧化应激影响NO来参与AM的发病过程。
Objective To investigate the expression of 12-lipoxygenase (12-LO), the level of NO in peripheral blood and its relationship with apoptosis in experimental autoimmune myocarditis (EAM) mice. Methods Male BALB / C mice (4 to 5 weeks old) were randomly divided into immunized myocarditis group (AM group) and normal control group (C group). In the AM group, 0.2 ml mixed solution containing 100 μg porcine myoglobin was subcutaneously injected in the groin on day 1,7 of the experiment. Group C was injected with the same amount of mixture (excluding pig cardiac myoglobin) in the same site at the same time. On the 14th, 21st and 30th days of the experiment, 8 mice in each group were randomly selected for blood sampling and sacrificed. Cardiac specimens were collected. The levels of 12-LO mRNA and iNOS mRNA in mouse cardiomyocytes were measured by RT-PCR. The level of NO was detected by nitrate reductase method. The apoptosis index (AI) was detected by TUNEL. Results The levels of 12-LO mRNA, iNOS mRNA, NO and AI in AM group were significantly higher than those on the 14th day on the 21st day (1.53 ± 0.27; 92.74 ± 3.99; 76.44 ± 3.01; 24.97 ± 1.54, respectively) 0.28; 82.99 ± 2.64; 57.51 ± 2.17; 13.20 ± 2.54) and 30 days (1.05 ± 0.10; 87.40 ± 3.32; 64.77 ± 3.92; 14.95 ± 2.15, respectively) The above indexes were significantly increased at the time point (all P <0.05). There was a significant positive correlation between 12-LO mRNA and NO and AI in AM group (r = 0.882; 0.877, P <0.05). Conclusions The expression of 12-LO increased in cardiomyocytes of autoimmune myocarditis and promoted the apoptosis of cardiomyocytes and progression of the disease. 12-LO may be involved in the pathogenesis of AM through the effect of oxidative stress on NO.