缺氧后处理对糖尿病性心肌细胞缺氧复氧时保护作用削弱的机制：与DJ-1表达的关系  

Mechanism of reduction of protection induced by hypoxic postconditioning in diabetic myocardiocytes subjected to hypoxia-reoxygenation： the relationship with D J-1 expression

作　　者：周斌[1] 夏中元[1] 赵博[1] 孙倩[1] 薛锐[1] 刘敏[1] 

机构地区：[1]武汉大学人民医院麻醉科,430060

出　　处：《中华麻醉学杂志》2016年第4期495-497,共3页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金（81471844）

摘　　要：目的评价缺氧后处理对糖尿病性心肌细胞缺氧复氧时保护作用削弱的机制及其与DJ-1表达的关系。方法正常培养的H9e2细胞，采用随机数字表法分为6组（n=36）：对照组（C组）、缺氧复氧组（H／R组）、缺氧后处理组（HPO组）、质粒转染DJ．1基因组（D组）、质粒转染DJ-1基因＋缺氧复氧组（DH组）和质粒转染DJ-1基因＋缺氧复氧＋缺氧后处理组（DHH组）。采用缺氧4h复氧2h的方法制备心肌细胞缺氧复氧损伤模型。C组、H／R组和HPO组采用高糖（30mmol／L）培养基培养H9c2细胞48h，H／R组和HPO组制备模型，HPO组复氧前行3个循环的复氧5min，缺氧5min处理。D组、DH组和DHH组H9c2细胞质粒转染DJ-1基因（pEX-2-EGFP—DJ-1），随后处理对应以上3组。于复氧2h时，采用CCK-8试剂盒检测细胞存活率，ELISA法检测上清液LDH水平，电镜下观察自噬小体，Western blot法检测细胞DJ-1、p62表达及LC3Ⅱ／Ⅰ比值。结果与c组比较，H／R组和HPO组细胞存活率、DJ-1表达水平降低，LDH活性升高（P〈0．01）；H／R组与HPO组比较上述指标差异无统计意义（P〉0．05）。与D组比较，DH组细胞存活率降低，LDH活性升高（P〈0．01）；与DH组比较，DHH组细胞存活率、自噬小体数量、LC3Ⅱ／Ⅰ比值升高，LDH活性和p62表达水平降低（P〈0．01）；H／R组与DH组比较上述指标差异无统计学意义（P〉0．05）。结论缺氧后处理对糖尿病性心肌细胞缺氧复氧时保护作用削弱的机制与高糖抑制DJ-1表达，降低自噬有关。Objective To evaluate the mechanism of reduction of protection induced by hypoxic postconditioning （HPO） in diabetic myocardiocytes subjected to hypoxia-reoxygenation （H/R） , and the relationship with DJ-1 expression. Methods H9c2 cells cultured in normal culture atmosphere were randomly divided into 6 groups （n=36 each） using a random number table： control group （group C） , group H/ R, group HPO, plasmid carrying DJ-1 gene transfeetion group （group D）, plasmid carrying DJ-1 gene transfection ＋ H/R group （group DH）, and plasmid carrying DJ-1 gene transfection ＋ H/R ＋ HPO group （group DHH）. H/R was induced by 4 h of hypoxia followed by 2 h of reoxygenation. H9c2 cells were cultured in high glucose culture medium （30 mmol/L） for 48 h in C, H/R and HPO groups. H/R model was established in H/R and HPO groups. HPO was induced by 3 cycles of 5 rain reoxygenation and 5 rain hypoxia before the onset of reoxygenation in group HPO. In D, DH and DHH groups, the plasmid carrying DJ-1 gene （pEX-2-EGFP-DJ-1） was transfected into the cells, and then the cells were cultured in high glucose culture medium and subjected to H/R and HPO, respectively. At 2 h of reoxygenation, the cell surviral rate was measured using the cell counting kit-8 assay, and the level of lactate dehydrogenase （LDH） in the supernatant was detected by enzyme-linked immunosorbent assay, the autophagosomes were examined with a transmission electron microscope, and the expression of D J-1 and p62 and ratio of microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ （LC3 Ⅱ/Ⅰ） in cells were detected by Western blot. Results Compared with group C, the cell survival rate and DJ-1 expression were significantly decreased, and the LDH activity was significantly increased in H/R and HPO groups （P〈0.01）. There was no significant difference in the parameters mentioned above between H/R and HPO groups （P〉0.05）. Compared with group D, the cell survival rate was significantly decreased, and the LDH activity w

关 键 词：缺氧 缺血后处理 糖尿病 肌细胞 心脏 再灌注损伤 细胞内信号肽和蛋白质类 

分 类 号：R614[医药卫生—麻醉学]