逆转录病毒介导的HO-1基因在尼洛替尼诱导K562／A02耐药细胞凋亡中的作用

The effect of retrovirus-mediated HO-1 gene on chronic myeloid leukemia resistance cell K562/A02 apoptosis induced by nilotinib

目的研究逆转录病毒介导的血红素加氧酶-1（HO-1）基因在尼洛替尼（Nilotinib，AMN107）诱导慢性髓性白血病（CML）耐药细胞凋亡中的作用。方法制备高病毒滴度的逆转录病毒载体pQCXIP-EGFP-HO-1，建立稳定转染HO-1基因的K562／A02细胞。采用RT-PCR鉴定K562／A02细胞中HO-1基因的表达。liT-PCR和Westernblot法检测AMN107作用24h后K562／A02细胞中HO-1mRNA和蛋白的表达，采用MTT法观察细胞增殖变化，通过实时荧光定量PCR（RQ-PCR）法检测bcr-abl融合基因的表达，通过流式细胞术检测细胞凋亡和细胞周期分布。结果成功构建重组逆转录病毒载体，并建立稳定转染HO-1基因的K562／A02细胞系；证实转基因组细胞HO-1mRNA显著表达。AMN107作用3组细胞后，转基因组细胞HO-1mRNA和蛋白的表达明显高于转空载体组和未转染组，差异有统计学意义（P〈0．05）；MTY法检测显示AMN107处理后细胞增殖受抑，而转基因组细胞存活率明显高于转空载体组和未转染组，差异有统计学意义（P〈0．05）；RQ-PCR法检测结果显示，10μmol／LAMN107抑制bcr-abl基因的表达，但转基因组的bcr-abl基因表达水平（Ct值18．15±0．18）高于转空载体组（20．32±0．20）和未转染组（20．51±0．21），差异有统计学意义（P〈0．05）；流式细胞术检测结果显示10ixmol／LAMN107作用24h后，转基因组、转空载体组、未转染组细胞凋亡率分别为（17．26±0．23）％、（39．47±0．17）％、（41．84±0．09）％，未加药转基因组、未加药未转染组细胞凋亡率分别为（3．74±0．03）％、（5．91±0．08）％；细胞周期分析结果显示经AMN107处理后，G0／G1期和S期细胞明显减少，细胞阻滞在G2／M期，而转基因细胞组细胞周期改变不如转空载体组、未转染组明显。结论AMN107可抑制CML耐药细胞增殖且诱导细胞凋亡，HO-1基因在CML耐药细胞凋亡过程中起保护作用，与促进CML耐药细胞的生长有关。

Objective To investigate the effect of retrovirus mediated heme oxygenase （HO）-1 gene on chronic myeloid leukemia（CML） resistance cell apoptosis induced by nilotinib （AMN107）. Methods High titer viral particles of pQCXIP-EGFP-HO-1 were prepared, and K562/A02 cells stably transfected with HO-1 gene was established. The expression of HO-1 in K562/A02 cells was detected by RT-PCR. After trea- ted with AMN107 for 24 h, HO-1 mRNA and protein expression by RT-PCR and Western blot, respectively; Cell proliferation by MTT assay; bcr-abl fusion gene by RQ-PCR, and the apoptosis and cell cycle by flow cy- tometry. Results Recombinant retrovirus vector was constructed successfully and K562/A02/HO-1 cells were successfully set up. The expression of HO-1 in K562/A02 cells was expressed clearly. After three groups cells treated with AMN107 for 24 h, the expression of HO-1 mRNA and protein was significantly high- er in gene-transfected group than in either empty vector or no-transfected group. The difference was statisiti- cally significant（P 〈0.05）. The cell proliferation was inhibited, but the cell viability was significantly higher in gene-transfected group than in other two groups. The difference was statistically significant （ P 〈 0.05 ） ;After treated with 10μmol/L AMN107 for 24 h, the Ct values of bcr-abl fusion gene were （18.15 ±0.18） in K562/A02/HO-1 group, being significantly higher than that in K562/A02/LXSN （ 20.32 ± 0. 20 ） and K562/A02 （20.51± 0.21 ） group, the difference was statistically significant （P 〈 0.05 ） ; the apoptosis rate were （ 17.26 ± 0.23 ） %, （ 39.47 ± 0. 17 ） %, and （41.84 ± 0.09 ） %, respectively in three groups, and were （3.74 ±0.03） %, （5.91±0.08） % in K563/A02/HO-1 untreated with drug and K562/A02 untreated with drug group. The number of Go/G1 phase and S phase cells markedly decreased. The cells were arrested in GJM phase. But cell cycle in gene-transfected group did not change significantly. Conclusion AMN107 inhibits proliferation of CML resistance cells and induces cell apoptosis. HO-1 gene can protect CML resist- ance cells to apoptosis. There was a relationship between HO-1 gene and the growth of CML resistance cells.