【作者】 王锋；

【导师】 刘季春；

【作者基本信息】 南昌大学 ， 外科学， 2009， 硕士

【摘要】 目的:以原代培养的SD新生乳鼠心肌细胞为研究对象,建立心肌细胞缺氧/复氧损伤模型,在细胞水平模拟心肌缺血再灌注损伤,探讨表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)预处理对心肌细胞缺氧/复氧(Anoxia/Reoxygenation,A/R)损伤的保护作用及其与细胞内PI3K-Akt信号转导通路的关系。方法:实验用SD新生大鼠(1～3 d),雌雄不拘,无菌取出乳鼠心脏,经分离附着组织,胰蛋白酶消化,纯化制成心肌细胞,在培养48 h后随机分为4组:正常对照(Control)组、缺氧/复氧(A/R)组、EGCG预处理组(EGCG+ A/R组)、EGCG+ LY294002+ A/R组。对以下指标进行检测:①细胞存活率(MTT法);②培养液中LDH活性;③流式细胞仪检测心肌细胞凋亡率;④Western blot法检测心肌细胞内t-Akt和p-Akt蛋白含量的表达。结果:①细胞存活率:A/R组(45.79±3.98%)与Control组(93.62±3.01%)、EGCG+ A/R组(73.86±4.78%)相比,有显著性差异(均p<0.01);EGCG+ LY294002+ A/R组(49.62±4.83%)与A /R组相比,无显著性差异(p>0.05),但与EGCG+ A/R组相比有显著性差异(p<0.01)。②培养液中LDH活性:A/R组(31.96±3.12 U/L)与Control组(4.06±0.51 U/L)比较,有显著差异(p<0.01);EGCG + A/R组(17.46±1.91 U/L)与A/R组比较,有显著性差异(p<0.01);EGCG+ LY294002+ A/R组(29.52±2.62 U/L)与A/R组相比,无显著性差异(p>0.05);但与EGCG +A/R组相比有显著性差异(p<0.01)。③心肌细胞凋亡率:与Control组(5.24±0.86%)、EGCG+ A/R组(9.68±1.12%)相比,A/R组(19.32±1.54%)心肌细胞凋亡程度明显增加,有显著性差异(均p<0.01);EGCG+ LY294002+ A/R组(17.62±1.48%)与A/R组相比,无显著性差异(p>0.05),与EGCG+ A/R组相比有显著性差异(p<0.01)。④心肌细胞内t-Akt及p-Akt表达含量的变化:各处理组心肌细胞内t-Akt蛋白的表达无显著性差异(均p>0.05)。p-Akt蛋白的表达在EGCG +A/R组中较A/R组显著增加,两者相比有显著性差异(p<0.01);EGCG+ LY294002+ A/R组心肌细胞内p-Akt蛋白的表达较EGCG +A/R组则明显降低,两者有显著性差异(p <0.01)。结论:从细胞水平证明了EGCG预处理对乳鼠心肌细胞缺氧/复氧损伤具有保护作用,这种预适应保护作用可能与EGCG激活了细胞内PI3K-Akt信号转导通路有关。更多还原

【Abstract】 Objective: Establish neonatal rat myocardial cells anoxia/reoxygenation (A/R) injury model. To explore the protective effects and the mechanisms of epigallocatechin gallate (EGCG) preconditioning on neonatal rat myocardial cells A/R Injury. Methods: Cultured myocardial cells of SD neonatal rats(1～3 d), and then randomly divided into three groups after cultured 48 hours: control group, anoxia/ reoxygenation group (A / R group) , EGCE preconditionging group(EGCG+ A / R group). At the end of experiment , measured cell viability and the amount of released lactate dehydrogenase (LDH) by a colorimetric method; Apoptosis of myocardial cells was detected by Annexin-V/PI assays with flow cytometry; The expression of t-Akt and p-Akt protein in cytoplasm were measured by western blotting. Results : 1. The cell viability: the cell viability of A/R group (45.79±3.98%) significantly decreased when compared with that of control group (93.62±3.01%) and EGCG+ A/R group (73.86±4.78%)(p<0.01, respectively); The viability of EGCG+ LY294002+ A/R group(49.62±4.83%)was no significant difference compared with that of A/R group respectively(p>0.05), While the difference between that of EGCG+ A/R group was significant(p<0.01). 2. LDH activity: the activity of LDH in A/R group(31.96±3.12 U/L)significantly increased compared with that in control group(4.06±0.51 U/L()p<0.01);the activity of LDH in EGCG + A/R group (17.46±1.91 U/L) significantly decreased compared with that in A/R group(p<0.01); the activity of LDH was no significant difference between EGCG+ LY294002+ A/R group(29.52±2.62 U/L)and A/R group (p>0.05), While the difference between that of EGCG+ A/R group was significant(p<0.01). 3. Cell apoptosis levels: the apoptosis levels of A/R group(19.32±1.54%)noticeably increased compared that of control group(5.24±0.86%)and EGCG+ A/R group(9.68±1.12%)(p<0.01, respectively); the apoptosis levels of EGCG+ LY294002+ A/R group(17.62±1.48%)was no significant difference compared with A/R group, While compared with EGCG+ A/R group, the the apoptosis levels was significantly decreased(p<0.01). 4. Expression of t-Akt and p-Akt protein: the expression of t-Akt protein was no noticeably difference in all group (p>0.05,respectively). The expression of p-Akt protein in EGCG+ A/R group was significantly increased compared that of A/R group(p<0.01); while the expression of p-Akt protein in EGCG+ LY294002+ A/R group was significantly decreased compared with EGCG+ A/R group(p<0.01). Conclusion: Our research showed that EGCG could protect cardiomyocytes against A/R injury, the antagonist effect on myocardial cells injury induced by A/R was mediated by activation of intracellular PI3K-Akt signal pathway.更多还原