吡柔比星对大鼠心肌细胞的损伤作用及其模型建立

doi:10.13481/j.1671-587x.20200509

摘要/Abstract

摘要： 目的：探讨吡柔比星（THP）对大鼠心肌细胞的损伤作用，阐明THP诱导心肌细胞损伤模型的建立方法。方法：常规体外培养大鼠心肌细胞H9C2，将细胞分为空白对照组和不同浓度（1×10^-6 mol·L^-1、1×10^-5 mol·L^-1、1×10^-4 mol·L^-1、1×10^-3 mol·L^-1和1 μmol·L^-1、3 μmol·L^-1、5 μmol·L^-1、7 μmol·L^-1、9 μmol·L^-1和10 μmol·L^-1 THP组，采用不同浓度THP处理细胞，并在6、12、24、36和48 h时间点采用CCK-8法检测各组细胞存活率，DCFH-DA活性氧（ROS）探针法检测细胞中ROS水平，硫代巴比妥酸比色法检测各组细胞中丙二醛（MDA）水平，黄嘌呤氧化法检测各组细胞中超氧化物歧化酶（SOD）活性，二硝基苯肼显色法检测各组细胞中乳酸脱氢酶（LDH）活性，TUNEL染色法检测各组细胞凋亡率，qRT-PCR法检测各组细胞中B淋巴细胞瘤-2相关X蛋白（Bax）、B淋巴细胞瘤2（Bcl-2）、半胱氨酸蛋白酶3（Caspase-3）和半胱氨酸蛋白酶9（Caspase-9）mRNA表达水平，Western blotting法检测各组细胞中Bax、Bcl-2、活化型Caspase-3（Cleaved Caspase-3）和活化型Caspase-9（Cleaved Caspase-9）蛋白表达水平。结果：与空白对照组比较，1、5和9μmol·L^-1 THP组H9C2细胞存活率降低（P<0.05或P<0.01），细胞中ROS和MDA水平及LDH活性升高（P<0.05或P<0.01），SOD活性降低（P<0.05），细胞凋亡率升高（P<0.05），细胞中Bax、Caspase-3和Caspase-9 mRNA表达水平升高（P<0.05或P<0.01），Bcl-2 mRNA表达水平降低（P<0.05），Bax、Cleaved Caspase-3和Cleaved Caspase-9蛋白表达水平升高（P<0.05或P<0.01），Bcl-2蛋白表达水平降低（P<0.01）。结论：THP对大鼠心肌细胞具有损伤作用，其中5 μmol·L^-1是THP诱导H9C2细胞损伤模型的最佳造模浓度。

关键词: 吡柔比星, 心肌细胞, 疾病模型,动物, 细胞凋亡, 细胞损伤

Abstract: Objective: To investigate the effect of pirarubicin (THP) on the cardiomyocyte injury in the rats, and to analyze the establishment method of THP-induced cardiomyocyte injury model. Methods: The rat cardiomyocytes H9C2 were routinely cultured in vitro. The cardiomyocytes H9C2 were divided into blank control group(0 μmol·L^-1 group) and different concentrations(1×10^-6 mol·L^-1, 1×10^-5 mol·L^-1, 1×10^-4 mol·L^-1, 1×10^-3 mol·L^-1, 1 μmol·L^-1, 3 μmol·L^-1, 5 μmol·L^-1, 7 μmol·L^-1, 9 μmol·L^-1,and 10 μmol·L^-1)of TNP groups. The H9C2 cells were treated with different concentrations of THP; at 6, 12, 24, 36 and 48 h, the survival rates of cells in various groups were detected by CCK-8 method; DCFH-DA reactive oxygen species (ROS) probe method was used to detect the intracellular ROS level; the malonaldehyde (MDA) levels in the cells in various groups were detected by thiobarbituric acid colorimetry, the superoxide dismntase(SOD) activities in the cells in various groups were detected by xanthine oxidation, and the lactic dehydrogenase(LDH) activities in the cells in various groups were detected by dinitrophenylhydrazine colorimetry; the apoptotic rates in various groups were determined by TUNEL staining; the expression levels of Bax, Bcl-2, Caspase-3 and Caspase-9 mRNA in the H9C2 cells in various groups were determined by qRT-PCR method;the expression levels of Bax, Bcl-2, Cleaved Caspase-3, and Cleaved Caspase-9 proteins in H9C2 cells in various groups were determined by Western blotting method. Results: Compared with blank control group, the survival rates of H9C2 cells in 1, 5 and 9 μmol·L^-1 THP groups were decreased (P<0.05 or P<0.01), the intracellular ROS and MDA levels and LDH activities were increased (P<0.05 or P<0.01), the SOD activities were decreased (P<0.05 or P<0.01), the apoptotic rates were increased(P<0.05), the expression levels of Bax, Caspase-3 and Caspase-9 mRNA were increased(P<0.05 or P<0.01), and the expression levels of Bcl-2 mRNA were decreased (P<0.01);the expression levels of Bax, Cleaved Caspase-3 and Cleaved Caspase-9 proteins were increased(P<0.05 or P<0.01), and the expression levels of Bcl-2 protein were decreased (P<0.01). Conclusion: THP has a damaging effect on the rat cardiomyocytes, of which 5 μmol·L^-1 is the optimal modeling concentration of THP-induced H9C2 injury model.

Key words: pirarubicin, cardiomyocytes, disease model,animal, apoptosis, cell injury

中图分类号:

R331

李琪, 徐瑞, 李藤藤, 江一川, 李敏. 吡柔比星对大鼠心肌细胞的损伤作用及其模型建立[J]. 吉林大学学报(医学版), 2020, 46(05): 948-954.

LI Qi, XU Rui, LI Tengteng, JIANG Yichuan, LI Min. Effect of pirarubicin on cardiomyocyte injury in rats and establishment of its model[J]. Journal of Jilin University(Medicine Edition), 2020, 46(05): 948-954.

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