环孢霉素A逆转人白血病耐药细胞系HL-60/ADM后氧自由基水平的变化

Change of Oxygen Free Radical in Reversing Multidrug-resistance of Human Leukemic Cell line HL-60/ADM by Cyclosporin A

本研究旨在探讨将抗阿霉素的人急性早幼粒细胞白血病细胞系HL-60/ADM的耐药性逆转后细胞内氧自由基水平的变化特点。选择环孢霉素A(CsA)作为耐药逆转剂,分别采用MTT法、流式细胞术和免疫组织化学法分析CsA对人白血病耐药细胞系的毒性及逆转效果;用化学比色法检测逆转前后细胞内丙二醛(MDA)、超氧化岐化酶(SOD)和谷胱甘肽(GSH)的含量。结果表明:当CsA在4μg/ml以下时对HL-60/ADM无明显毒性作用,超过此浓度,其毒性呈剂量效应(p<0.001)。当CsA浓度为0.5μg/ml时就有明显逆转作用,随着CsA剂量增加,逆转作用逐渐增强(p<0.001),当CsA剂量达8μg/ml以上时对细胞存活产生明显的影响。流式细胞仪检测细胞内药物浓度发现,逆转耐药细胞系HL-60/ADM+CsA细胞内阿霉素的浓度明显高于耐药细胞系HL-60/ADM。免疫组织化学检测结果显示,HL-60/ADM细胞膜上P-gp高表达,经CsA作用后P-gp表达下降。氧自由基测定结果显示:HL-60/ADM细胞经4μg/ml CsA作用72小时后细胞内SOD的活性与对照组相比显著降低(p<0.001),细胞内的脂质过氧化产物MDA的含量与对照组相比有所升高(p<0.05),抗氧化剂GSH的含量较对照组明显降低(p<0.001)。结论:CsA能有效逆转HL-60/ADM的耐药性;逆转后细胞内氧自由基的含量升高,抗氧化剂的活性明显减低,过多的氧自由基可影响细胞的功能状态,导致细胞死亡。

This study was aimed to evaluate the reversed effects of cyclosporin A （CsA） on multidrug resistance （MDR） of human leukemic cell line HL-60/ADM, and to investigate the relationship of the oxygen free radical content between HL-60/ADM cells and the reversed HL-60/ADM cells （ HL-60/ADM ＋ CsA ）. The cytotoxicity and the reversed effects of CsA on multidrug resistance of human leukemic cell line HL-60/ADM were studied by MTT, flow cytometry （FCM） and immunohistochemical assay; the oxygen free radical for HL-60/ADM and HL-60/ADM ＋ CsA cell lines were detected by colorimetric method. The results showed that the CsA less than 4 μg/ml had no significant cytotoxicity on HL-60/ADM, while the cytotoxicity was rised with CsA concentration increasing; And CsA （4 μg/ml） combined with ADM （ 1 μg/ml ） could obviously restrain the growth of HL-60/ADM cells （ p 〈 0. 001 ）. The P-gp expression of HL-60/ADM decreased obviously after exposure to CsA （4 μg/ml ） for 72 hours, at the same cell conditions, MDA concentration of the reversed groups （ HL-60/ADM ＋ CsA cells） was higher than that of the control groups （HL-60/ADM cells） （p 〈0.05）, while the levels of SOD and GSH in the reversed groups were significantly lower than that in control groups （p 〈 0.001 ）. It is concluded that MDR of HL-60/ADM can be reversed effectively by low dose of CsA, the level of oxygen free radical increases and the activity of antioxidants decreases in the reversed cells. Oxygen free radicals may be involved in this reverse process, which thereby lead to the cell death.