两种H9C2心肌细胞氧化损伤模型的比较  

Comparison of two models of oxidative damage in H9C2 cardiomyocytes

作　　者：陈芸霞 邓洪荣 刘蕙文 许皓[2] 易勤[1] 谭彬[1] 田杰[3] 朱静[1] Cheng Yunxia;Deng Hongrong;Liu Huiwen;Xu Hao;Yi Qin;Tan Bin;Tian Jie;Zhu Jin(Department of Pediatric Research Institute,Children’s Hospital of Chongqing Medical University,National Clinical Research Center for Child Health and Disorders,Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of Pediatrics;Department of Clinical Laboratory,Children’s Hospital of Chongqing Medical University;Department of Cardiology(Internal Medicine),Children’s Hospital of Chongqing Medical University)

机构地区：[1]重庆医科大学附属儿童医院儿研所、国家儿童健康与疾病临床医学研究中心、儿童发育疾病研究教育部重点实验室、儿科学重庆市重点实验室,重庆400014 [2]重庆医科大学附属儿童医院检验科,重庆400014 [3]重庆医科大学附属儿童医院心血管科(内科),重庆400014

出　　处：《重庆医科大学学报》2024年第9期1079-1085,共7页Journal of Chongqing Medical University

基　　金：国家自然科学基金面上资助项目(编号:82270271)。

摘　　要：目的:探讨低氧、低糖及血清剥夺(glucose and serum deprivation under hypoxia(1%O_(2)),GSDH)处理与H_(2)O_(2)处理在构建H9C2心肌细胞氧化损伤模型中的应用价值。方法:培养H9C2心肌细胞,当细胞生长状态良好时,用低氧(1%O_(2))、低糖(1.0 g/L)及血清剥夺联合处理或200μmol/L的H_(2)O_(2)作用于心肌细胞24 h。采用CCK8实验检测细胞增殖能力;通过细胞凋亡试剂(annexinV-FITC/PI)、Hoechst染色检测细胞凋亡;细胞活性氧(reactive oxygen species,ROS)检测细胞氧化应激水平;BODIPY检测细胞脂质过氧化水平;过碘酸雪夫(periodic acid-schiff stain,PAS)染色检测细胞糖原合成能力;线粒体膜电位检测试剂(mitochondrial membrane potential assay kit with JC-1,JC-1)染色检测线粒体膜电位水平;Western blot检测能量代谢相关分子AMP依赖的蛋白激酶(Adenosine 5’-monophosphate(AMP)-activated protein kinase,AMPK)及氧化应激相关分子NAD(P)H醌脱氢酶1(NAD(P)H quinone dehydrogenase 1,NQO-1)、血红素氧合酶1(heme oxygenase-1,HO-1)蛋白表达水平。结果:与空白组比较,GSDH处理组与H_(2)O_(2)处理组的心肌细胞存活率均降低,差异均有统计学意义(P<0.05)。与空白组相比,GSDH处理与H_(2)O_(2)处理都增加了心肌细胞的凋亡水平、ROS和脂滴堆积水平增高、糖原消耗量增加且线粒体膜电位降低。但相比于H_(2)O_(2)处理组,GSDH处理组的细胞糖原消耗增多更明显,脂滴堆积也更明显,并且AMPK磷酸化水平显著降低。结论:低氧、低糖及GSDH处理与H_(2)O_(2)处理均能造成H9C2心肌细胞氧化损伤,但相比于H_(2)O_(2)处理组,GSDH处理组的效果更符合体内心肌损伤时的能量代谢转变,有望作为一种更简单便捷的心肌氧化损伤模型应用于科研。Objective:To investigate the value of glucose and serum deprivation under hypoxia(1%O_(2))(GSDH)treatment and H_(2)O_(2)treatment in establishing a model of oxidative injury in H9C2 cardiomyocytes.Methods:H9C2 cardiomyocytes were cultured,and when the cardiomyocytes were in good growth conditions,they were treated with the combination of low oxygen(1%O_(2)),low glucose(1.0 g/L),and serum deprivation or H_(2)O_(2)200μmol/L alone for 24 hours.CCK8 assay was sued to measure the proliferation ability of cells;the apoptosis reagent(AnnexinV-FITC/PI)and Hoechst staining were used to measure cell apoptosis;reactive oxygen species(ROS)was used to measure the level of oxidative stress;BODIPY testing was used to measure the level of lipid peroxidation in cells;periodic acid-Schiff staining was used to measure the ability for glycogen synthesis;mitochondrial membrane potential assay kit with JC-1 staining was used to measure mitochondrial membrane potential;Western blot was used to measure the protein expression levels of the energy metabolism-related molecule AMP-activated protein kinase(AMPK)and the oxidative stress-related molecules NAD(P)H quinone dehydrogenase 1(NQO-1)and heme oxygenase-1(HO-1).Results:Compared with the blank group,both the GSDH treat⁃ment group and the H_(2)O_(2)treatment group had a significant reduc⁃tion in the viability of cardiomyocytes(P<0.05).Compared with the blank group,both GSDH treatment and H_(2)O_(2)treatment increased the levels of cardiomyocyte apoptosis,ROS and lipid droplet accumu⁃lation,and glycogen consumption,with a reduction in mitochondrial membrane potential in cardiomyocytes.However,compared with the H_(2)O_(2)treatment group,the GSDH treatment group showed significantly greater increases in glycogen consumption and lipid droplet accumulation and a significant reduction in AMPK phosphorylation.Conclusion:Both GSDH treatment and H_(2)O_(2)treatment can cause oxidative injury to H9C2 cardiomyocytes,but compared with H_(2)O_(2)treatment,the effect of GSDH treatment is more i

关 键 词：低氧低糖血清剥夺 过氧化氢 氧化应激 能量代谢 免疫印迹法 

分 类 号：R331[医药卫生—人体生理学]