心肌细胞培养方法改良及抗肿瘤药物心肌毒性评价

Improved culture approach of cardiomyocytes and evaluation of the cardiotoxicity of antitumor drugs

目的改良心肌细胞培养方法,评价抗肿瘤药物多柔比星(doxorubicin,DOX)、顺铂(cisplatinum,DDP)和氯法拉滨(clofarabine,CLO)的心肌细胞毒性。方法采用双酶依次消化法充分分离心脏组织中的心肌细胞,优化差速贴壁法进行细胞纯化的时长为20 min,台盼蓝染色法检测细胞成活率,免疫组织化学法鉴定心肌细胞纯度,MTS法分别测定多柔比星、顺铂和氯法拉滨对心肌细胞的毒性。结果较低浓度的多柔比星(0.17μmol·L-1)、顺铂(17.50μmol·L-1)和氯法拉滨(0.33μmol·L-1)即能引起与对照组细胞存在显著性差异(P<0.05),3种药物对心肌细胞的半数抑制浓度IC50值分别为(1.17±0.28)、(7.90±2.05)和(34.07±14.43)μmol·L-1。结论与各自对照组细胞相比,3种抗肿瘤药物的心肌细胞毒性呈浓度依赖性增加,且心肌细胞在药物暴露早期对其敏感性较高,提示临床需警惕药物引起的心脏毒性。

Objective To improve the culture approach of cardiomyocytes,and evaluate the cardiotoxicity of antitumor drugs of doxorubicin（ DOX）,cisplatinum（ DDP） and clofarabine（ CLO）. Methods Cardiomyocytes were isolated from heart tissue of neonatal rat by double-enzyme digestion method,and differential adhesion was optimized as 20 min to purify the newly isolated cardiomyocyts. Cell survival rate was tested by trypan blue stain assay,and the purity of cardiomyocytes was identified by immunohistochemical method. Afterward the cytotoxicity of DOX,DDP and CLO on cardiomyocytes were determined by MTS assay,respectively.Results Compared with correponding control group,significant differences were revealed at lower concentrations of DOX（ 0. 17 μmol·L- 1）,DDP（ 17. 50 μmol·L- 1） and CLO（ 0. 33 μmol·L- 1）（ P 0. 05）,respectively. The concentrations of DOX,DDP and CLO resulting in 50% viability of primary cardiomyocytes（ IC50） were（ 1. 17 ± 0. 28）,（ 7. 90 ± 2. 05） and（ 34. 07 ± 14. 43） μmol·L- 1,respectively.Conclusions Comparing with the control cell in each group,the cardiotoxicity of three antitumor drugs was increased in concentration-dependent manner,and cardiomyocytes are more sensitive to these drugs in the early phase of drug exposure,which suggests that the cardiotoxicity of antitumor drugs should be cautioned for clinical application.