多柔比星诱导体外培养心肌细胞损伤模型的建立

Establishment of toxic cardiomyocytes model induced by doxorubicin in vitro

目的探讨建立多柔比星(DOX)损伤心肌细胞模型的方法和可行性。方法原代培养4d的SD乳鼠心肌细胞,用不同浓度的DOX进行处理,MTT法测定不同时间点的心肌细胞存活率,倒置显微镜下观察心肌细胞搏动频率,比色法检测上清液中乳酸脱氢酶(LDH)和天门冬氨酸氨基转移酶(AST)的含量,Hoechst 33258荧光染色检测心肌细胞的凋亡情况。结果不同浓度的DOX对心肌细胞均有损伤。当DOX浓度大于1.0mg/L时,心肌细胞存活率明显下降。随着DOX浓度的增加,心肌细胞搏动频率减慢,LDH及AST明显增高,细胞凋亡增加,差异有统计学意义(P<0.05)。而随着DOX作用时间的延长,心肌细胞存活率亦显著降低。结论DOX对体外培养心肌细胞损伤程度呈浓度和时间依赖性,1.0mg/L的浓度作用4h可建立可靠的心肌细胞毒性损伤模型。

Objective To investigate the methods and feasibility to establish experimental model of toxic cardiomyocytes induced by doxorubicin（DOX）. Methods The primarily cultured neonatal SD rat cardiomyocytes were exposed to different concentrations of DOX and analyzed at different post-treatment durations after having been cultured for 4d. The cardiomyocytes viability was determined by MTT assay. The beating rate of cardiomyoeytes was detected by inverted microscope. The activity of lactic dehydrogenase （LDH） and aspartate aminotransferase （AST） in superior fluid were measured by colorimetry. The cardiomyocyte apoptosis was detected by Hoechst 33258 staining. Results DOX had injury effect on cardiomyocytes with different concentration. The survival rate decreased significantly when the concentration was above 1.0mg/L. Accompanied with the DOX concentration increased, the beating rate of cardiacmyocytes decreased, and the activity of LDH and AST increased, the apoptosis rate increased significantly, compared with the control group（P〈0.05）. Meantime, the longer the time DOX contacted with cardiacmyocytes, the lower the survival rate was. Conclusion The damage on cardiomyocytes induced by DOX is dose-dependent and time-dependent. The toxic cardiomyocytes model can be established when primary cultured eardiomyocytes are exposed to 1.0mg/L doxorubiein for 4h.