摘要
目的 评价PTEN在糖尿病因素削弱缺氧后处理对心肌细胞保护效应中的作用及其与糖原合成酶激酶3β（GSK-3β）介导的线粒体凋亡途径的关系.方法 高糖培养（30 mmol/L） 24 h的H9c2细胞,采用随机数字表法分为6组（n=5）：常氧组（N组）、缺氧复氧组（H/R组）、缺氧后处理组（HPO组）、PTEN基因沉默常氧组（P-N组）、PTEN基因沉默缺氧复氧组（P-H/R组）和PTEN基因沉默缺氧后处理组（P-HPO组）.H9c2细胞置于培养箱（95％N2＋5％CO2）孵育4h再置于常氧培养箱（90％ O2＋ 10％ CO2）孵育2h建立缺氧复氧模型.于复氧前行3个循环的复氧5 min/缺氧5 min进行缺氧后处理.于复氧末,采用ELISA法检测上清液LDH水平,JC-1荧光法测定线粒体膜电位的变化,AnnexinV-FITC/PI流式细胞术检测细胞凋亡率,Western blot法检测PTEN和磷酸化GSK-3β（p-GSK-3β）的表达.结果 与N组比较,H/R组和HPO组LDH释放量、JC-1单聚体/多聚体比值和细胞凋亡率增加,PTEN表达上调（P＜0.05）;H/R组与HPO组上述指标比较差异无统计学意义（P＞0.05）;与HPO组比较,P-HPO组LDH释放量和JC-1单聚体/多聚体比值和细胞凋亡率降低,PTEN表达下调,p-GSK-3β表达上调（P＜0.05）.P-N组与N组比较,P-H/R组与H/R组比较,PTEN表达下调,其他指标差异无统计学意义（P＞0.05）.结论 PTEN参与了糖尿病因素削弱缺氧后处理对心肌细胞的保护效应,机制与其激活GSK-3β调控的线粒体凋亡途径有关.
Objective To evaluate the role of phosphatase and tensin homologue deleted on chromosome 10 （PTEN） in diabetes mellitus-induced reduction of hypoxic postconditioning （HPO）-induced protection of cardiomyocytes and the relationship with glycogen synthase kinase-3β （GSK-3β）-mediated mitochondrial apoptotic pathway.Methods H9c2 cells incubated in high-glucose （30 mmol/L） medium for 24 h were divided into 6 groups （n =5 each） using a random number table：normoxia group （group N）,hypoxia-reoxygenation （H/R） group,group HPO,PTEN gene silencing normoxia group （group P-N）,PTEN gene silencing H/R group （group P-H/R）,and PTEN gene silencing HPO group （group P-HPO）.H9c2 cells were exposed to 95% N2-5% CO2 for 4 h followed by 2 h reoxygenation with 90% O2-10% CO2.HPO was induced by 3 cycles of 5 min reoxygenation followed by 5 min hypoxia before reoxygenation.At the end of reoxygenation,the level of lactate dehydrogenase （LDH） in the supernatant was detected by enzyme-linked immunosorbent assay,the changes in mitochondrial membrane potential （MMP were assessed by JC-1 fluorescence assay,the cell apoptosis was detected by AnnexinV-FITC/PI flow cytometry,and the expression of PTEN and phosphorylated GSK-3β （p-GSK-3β） was determined by Western blot.The JC-1 monomer/polymer ratio and apoptosis rate were calculated.Results Compared with group N,the amount of LDH released,JC-1 monomer/polymer ratio and apoptosis rate were significantly increased,and the expression of PTEN was up-regulated in H/R and HPO groups （P〈0.05）.There was no significant difference in the parameters mentioned above between group H/R and group HPO （P〉0.05）.Compared with group HPO,the amount of LDH released,JC-1 monomer/polymer ratio and apoptosis rate were significantly decreased,PTEN expression was down-regulated,and the expression of p-GSK-3β was up-regulated in group P-HPO （P〈0.05）.Compared with group N,the expression of PTEN was significantly down-regulated,and no significant changes
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2017年第1期108-111,共4页
Chinese Journal of Anesthesiology
基金
国家自然科学基金（81471844,81671891）
关键词
糖原合成酶激酶3
缺氧
糖尿病
肌细胞
心脏
再灌注损伤
后处理
PTEN
Glycogen synthase kinase 3
Anoxia
Diabetes mellitus
Myocytes, cardiac
Reperfusion injury
Post-conditioning
PTEN
