HO-1基因表达对伊马替尼耐药的慢性髓系白血病细胞增殖的影响

Effects of HO-1 gene expression on proliferation of imatinib resistant CML cells

目的通过氯高血红素（Heroin）诱导伊马替尼耐药慢性髓系白血病（CML）细胞株K562／A02．IM中血红素加氧酶-1（HO-1）基因表达，探讨HO-1基因对伊马替尼耐药CML细胞增殖的影响，为治疗CML多药耐药及新药的开发提供思路和实验依据。方法采用RT—PCR法检测20例CML伊马替尼耐药患者骨髓细胞中HO-1基因的表达，半定量RT—PCR法和Westernblot法分别检测不同剂量Hemin处理K562／A02-IM细胞不同时间后HO-1的表达，通过AnnexinV／H双染色法检测细胞凋亡情况，采用MTT法检测Hemin诱导及锌原卟啉抑制HO-1表达与细胞存活率的关系。结果RT—PCR结果显示耐药患者骨髓细胞中HO-1基因阳性表达，半定量RT—PCR和Westernblot法显示，不同浓度的Heroin（0、10、20及40μmol／L）处理K562／AO2-IM细胞16h后，HO-1的表达量随Heroin浓度的升高而增加，存在剂量依赖关系，而20μmol／LHeroin分别处理K562／AO2-IM细胞0、8、16、24h后，HO-1的表达量在处理16h组最高。AnnexinV／PI法检测显示，0、10、20及40pmlol／LHemin作用于K562／AO2-IM细胞16h后细胞凋亡率分别为（17．61±0．01）％、（12．13±0．11）％、（7．94±0．03）％和（4．62±0．15）％，其抗凋亡的作用呈剂量依赖性；20pmol／LHemin作用K562／A02-IM细胞8、16及24h的细胞凋亡率分别为（14．72±0．05）％、（8．15±0．07）％和（16．37±0．13）％。MTT法检测显示与对照组相比，Hemin诱导K562／AO2-IM细胞HO-1基因表达促进了细胞的增殖，且作用存在剂量依赖关系；而锌原卟啉抑制HO-1的表达，促进了细胞的凋亡（P〈0．05）。结论CML伊马替尼耐药患者骨髓细胞HO-1基因阳性表达，HO-1是一种可诱导表达型基因，具有抗细胞凋亡及促进细胞增殖的作用，抑制HO-1表达可能成为治疗CML耐药的新方法。

Objective To investigate the effect of heine oxygenase-1 （ HO-1 ） expression on cell growth and apoptosis in imatinib resistant chronic myeloid leukemia （CML） cells （K562/A02-IM） , and explore the relationship between HO-1 gene and CML. Methods The expression of HO-1 in 20 drug-resistant CML patients was detected by RT-PCR. Different concentrations of heroin were used to induce HO-1 expres- sion of K562/A02-IM, HO-1 expression at different time was detected by RT-PCR and Western blot analysis. Cell apoptosis was detected by Annexin V/PI staining, and MTT assay was used to detect viability of K562/ A02-1M cells after induction or inhibition of HO-1 gene by heroin and zinc protoporphyrin （ZPP）. Results RT-PCR showed that HO-1 was expressed in the bone marrow mononuclear cells （BMMNCs）. When treated with heroin at different concentrations （0, 10, 20, 40 μmol/L） for 16 h, the expression of HO-1 in K562/ AO2-IM was increased in a dose-dependent manner, and peaked at 20 μmot/L of hemin for 16 h. The apoptosis rates were （17.61 ±0.01）% , （12.13±0.11）% , （7.94 ±0.03）% and （4.62±0.15）% at 0,10, 20 and40 Ixmol/L of hemin respectively for 16 h and were （14.7±0.05）% , （8. 1±0.07）% and （16.3 ± 0.13）% at 20μmol/L of hemin treatment for 8,16, and 24 h respectively. Hemin induced apoptosis of K562/AO2-IM cells in a dose-dependent manner. The expression of HO-I was induced in K562/AO2-IM cells in a dose-dependent manner, and the survival of K562/AO2-IM cells was significantly increased as compared to that of control group. When HO-1 was inhibited by ZPP, the ceils survival was sharply decreased compared to that of the control group （P 〈0.05）. Conclusion HO-1 was expressed in the BMMNCs. It is a kind of molecules whose expression can be induced and can promote the growth of drug-resistant cells. Inhibition of HO-1 expression probably be used for the treatment of drug-resistant CML.