摘要
目的通过原代培养SD大鼠的乳鼠心肌细胞建立H2O2心肌细胞氧化应激损伤模型,观察脂联素对心肌细胞氧化应激所致内质网应激的保护作用。方法采用酶消化法原代培养乳鼠心肌细胞,倒置相差显微镜下观察细胞生长状态,通过α-肌动蛋白免疫荧光法对培养的心肌细胞进行鉴定。选用原代培养3~4天的心肌细胞,随机分为对照组、H2O2组、H2O2＋10 mg/L脂联素组、H2O2＋20 mg/L脂联素组和H2O2＋30 mg/L脂联素组。实验终止后,在倒置相差显微镜下观察心肌细胞形态的变化,采用化学比色法测定乳酸脱氢酶的释放,通过流式细胞术来检测心肌细胞的凋亡,用RT-PCR与W estern B lotting方法检测内质网应激指标GRP78和Caspase-12的表达。结果与对照组相比,给予H2O2后,细胞凋亡率显著增加（70.7%±6.4%比1.0%±0.6%,P〈0.05）,LDH释放增加（1411.5±189.7 U/L比353.3±50.3 U/L,P〈0.05）,内质网伴侣蛋白GRP78以及Caspase-12在mRNA（分别为1.25±0.50比0.18±0.10和1.32±0.15比0.26±0.06）及蛋白水平（分别为0.92±0.50比0.37±0.10和1.24±0.50比0.51±0.01）表达增加（P〈0.05）,30 mg/L脂联素预处理后给予H2O2,可较大程度地逆转上述指标变化,细胞凋亡率显著下降（43.6%±3.8%）,LDH释放减少（686.7±61.1 U/L）,内质网伴侣蛋白GRP78以及Caspase-12在mRNA（分别为0.56±0.03和0.83±0.04）及蛋白水平（分别为0.66±0.03和0.64±0.03）表达减少（P〈0.05）。结论氧化应激使GRP78和Caspase-12表达增强,启动内质网应激,脂联素可以通过减轻内质网应激逆转H2O2所致的心肌细胞损伤及凋亡作用,对心肌细胞有保护作用。
Aim To investigate the effects of adiponectin on endoplasmic reticulum stress injury of the 3～4 days SD rat cardiomyocytes,which was induced by the intervention of H2O2.Methods Primary cardiomyocytes were obtained from neonatal rat and cultured by enzymatic digestion methods.The morphology of neonatal rat cardiomyocytes was studied by phase contramicroscope.Its molecular markers were observed by α-actin immunocytochemistry.Primary 3～4 days cells were used in experiment,and they were randomly divided into control group,H2O2 group（200 μmol/L H2O2,2 h）,H2O2＋10 mg/L APN group,H2O2＋20 mg/L APN group,and H2O2＋30 mg/L APN group.The change of morphology of cardiomyocytes was observed by electron microscope.The content of lactate dehydrogenase（LDH） was detected by chemistry chromatometry;the cardiomycocytes apoptosis was detected by agarose gel electrophoresislevels and Annexin V-FITC/PI flow cytometry.The expressions of GRP78 and caspase-12 were detected by Western Blotting and RT-PCR.Results Adiponectin pretreatment significantly decreased the apoptosis rate（43.6%±3.8% vs 70.7%±6.4%）,and the release of LDH（686.7±61.1 U/L vs 1411.5±189.7 U/L,P0.05）.The expression of GRP78 and caspase-12 attenuated（mRNA： 0.56±0.03 vs 1.25±0.50;0.83±0.04 vs 1.32±0.15）,（protein： 0.66±0.03 vs 0.92±0.50;0.64±0.03 vs 1.24±0.50）.Conclusion Oxidative stress enhances the expressions of GRP78 and caspase-12,and makes endoplasmic reticulum stress to start.Adiponectin which can attenuate endoplasmic reticulum stress,have a protective effect on myocardial cells.
出处
《中国动脉硬化杂志》
CAS
CSCD
北大核心
2009年第12期980-984,共5页
Chinese Journal of Arteriosclerosis
关键词
内质网应激
脂联素
心肌细胞
氧化应激
H2O2
Endoplasmic Reticulum Stress
Adiponectin
Cardiomyocytes
Oxidative Stress
H2O2
