摘要
目的探讨敲低蛋白酶激活受体2(PAR2)基因对缺氧/复氧(H/R)损伤后H9c2心肌细胞氧化应激的影响。方法将H9c2心肌细胞分为非特异性干扰组(NS组)和特异性干扰组(SI组),分别转染非特异小干扰RNA(siRNA)和PAR2 siRNA。转染48 h后检测两组PAR2 mRNA表达情况以评价PAR2敲除效率。取成功转染siRNA的H9c2细胞,建立H/R模型,采用化学比色法测定细胞培养液中乳酸脱氢酶(LDH)的含量,2',7'-二氯荧光黄双乙酸盐荧光探针检测细胞内活性氧(ROS)含量,黄嘌呤氧化酶法测定细胞超氧化物歧化酶(SOD)的活力,硫代巴比妥酸显色法测定细胞丙二醛的含量。结果转染48 h后,SI组PAR2 mRNA表达水平低于NS组(P<0.05),且降至NS组30%以下。SI组ROS相对荧光强度、丙二醛及LDH含量均低于NS组(均P<0.05),SOD活力高于NS组(均P<0.05)。结论 PAR2基因敲低可减轻损伤后H9c2心肌细胞的氧化应激,从而发挥心肌保护作用。
Objective To investigate the effect of knocking down protease-activated receptor 2(PAR2) gene on oxidative stress in H9c2 myocardial cells after hypoxia/reoxygenation(H/R) injury. Methods H9c2 myocardial cells were divided into non-specific interference(NS) group and specific interference(SI) group,with transfection by non-specific small interfering RNA(siRNA) and PAR2 siRNA,respectively,in the two groups.After 48 h of transfection,PAR2 mRNA expression was detected in the two groups to evaluate the efficiency of PAR2 knockout.The model of H/R was established following a successful siRNA transfection in H9c2 cells,then the content of lactate dehydrogenase(LDH) in the cell culture media was determined by chemical colorimetry,the content of intracellular reactive oxygen species(ROS) by 2′,7′-dichlorodihydrofluorescein diacetate fluorescent probe,the activity of superoxide dismutase(SOD) by xanthine oxidase method,and the content of malondialdehyde by thiobarbituric acid assay. Results After 48 h of transfection, the expression level of PAR2 mRNA in the SI group was lower than that in the NS group( P <0.05),and decreased to 30% of the level of the NS group.Compare with the NS group,the SI group exhibited lower relative fluorescence intensity of ROS,along with lower contents of malondialdehyde and LDH,and higher SOD activity(all P <0.05). Conclusion Knockdown of PAR2 gene can alleviate oxidative stress of H9c2 myocardial cells after injury,thus exerting a protective effect on myocardium.
作者
王敏
张珊
慕家盛
王媛媛
杨青男
黄泽旭
陈启稚
WANG Min;ZHANG Shan;MU Jia-sheng;WANG Yuan-yuan;YANG Qing-nan;HUANG Ze-xu;CHEN Qi-zhi(Department of Cardiovascular Medicine,Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China)
出处
《广西医学》
CAS
2019年第7期855-858,872,共5页
Guangxi Medical Journal
基金
国家自然科学基金(81300087)
上海市卫生系统重要疾病联合攻关项目(2014ZYJB0501)
关键词
蛋白酶激活受体2基因
氧化应激
缺氧/复氧损伤
H9C2心肌细胞
敲低
转染
Protease-activated receptor 2 gene
Oxidative stress
Hypoxia/reoxygenation injury
H9c2 myocardial cell
Knockdown
Transfection
