Chen Cheng, Wang Ji-shi, Qin Dong, Yang Yuan, Yu Yan-yan, Fang Qin
Department of Hematology, Affiliated Hospital of Gui Yang Medical College, Guiyang, China.
Zhonghua Xue Ye Xue Za Zhi. 2012 May;33(5):383-7.
To investigate the effect of retrovirus mediated heme oxygenase (HO)-1 gene on chronic myeloid leukemia (CML) resistance cell apoptosis induced by nilotinib (AMN107).
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 treated 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 cytometry.
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 higher in gene-transfected group than in either empty vector or no-transfected group. The difference was statistically significant (P < 0.05). The cell proliferation ofs 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 10 µmol/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 G(0)/G(1) phase and S phase cells markedly decreased. The cells were arrested in G(2)/M phase. But cell cycle in gene-transfected group did not change significantly.
AMN107 inhibits proliferation of CML resistance cells and induces cell apoptosis. HO-1 gene can protect CML resistance cells to apoptosis. There was a relationship between HO-1 gene and the growth of CML resistance cells.
探讨逆转录病毒介导的血红素加氧酶（HO）-1基因对尼罗替尼（AMN107）诱导的慢性髓性白血病（CML）耐药细胞凋亡的影响。
制备高滴度的pQCXIP-EGFP-HO-1病毒颗粒，建立稳定转染HO-1基因的K562/A02细胞。采用RT-PCR检测K562/A02细胞中HO-1的表达。用AMN107处理24 h后，分别采用RT-PCR和Western blot检测HO-1 mRNA和蛋白表达；采用MTT法检测细胞增殖；采用RQ-PCR检测bcr-abl融合基因；采用流式细胞术检测细胞凋亡和细胞周期。
成功构建重组逆转录病毒载体，成功建立K562/A02/HO-1细胞。K562/A02细胞中HO-1表达明显。三组细胞经AMN107处理24 h后，基因转染组HO-1 mRNA和蛋白表达明显高于空载体组和未转染组。差异有统计学意义（P<0.05）。细胞增殖受到抑制，但基因转染组细胞活力明显高于其他两组。差异有统计学意义（P<0.05）；用10 μmol/L AMN107处理24 h后，K562/A02/HO-1组bcr-abl融合基因的CT值为（18.15±0.18），明显高于K562/A02/LXSN组（20.32±0.20）和K562/A02组（20.51±0.21），差异有统计学意义（P<0.05）；三组凋亡率分别为（17.26±0.23）%、（39.47±0.17）%和（41.84±0.09）%，未用药的K563/A02/HO-1组和未用药的K562/A02组凋亡率分别为（3.74±0.03）%、（5.91±0.08）%。G(0)/G(1)期和S期细胞数量明显减少。细胞停滞于G(2)/M期。但基因转染组细胞周期无明显变化。
AMN107抑制CML耐药细胞增殖并诱导细胞凋亡。HO-1基因可保护CML耐药细胞免于凋亡。HO-1基因与CML耐药细胞生长之间存在关联。
