辛伐他汀联合全反式维甲酸对人早幼粒细胞白血病细胞株WT1／hDMP1基因表达的影响

Effects of simvastatin plus all-trans retinoic acid on WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4

目的探讨羟甲基戊二酸单酰辅酶A（HMG—CoA）还原酶的抑制剂辛伐他汀（SV）联合全反式维甲酸（ATRA）对人早幼粒细胞白血病NB4细胞株增殖、分化与凋亡的影响，以及肾母细胞瘤（WT1）基因／转录因子人细胞周期调节蛋白依赖性myb样蛋白1（hDMP1）基凶表达变化。方法以不同浓度SV联合ATRA处理NB4细胞，取对数生长期细胞进行实验。各组细胞分别进行细胞形态观察；四甲基偶氮唑盐（MTr）法观察细胞增殖能力；流式细胞仪测定NB4细胞分化指标CD11b和细胞凋亡指标膜联蛋白（Annexin—V）／碘化丙啶（PI）变化；实时（Real—time）RT—PCR测定WT1／hDMPI基因表达变化。结果15、10、5μmol／LSV单独处理NB4细胞，随着培养时间延长，细胞抑制率增加（F=7．15，P=0．000），CD11b的表达水平逐渐升高（F=3．41，P=0．014），Annexin—V表达水平逐渐增高（F=43．38，P=0．000），WT1基因表达水平逐渐降低（F=5．35，P=0．001），同时伴随着hDMP1基因表达水平的增加（F=22．61，P=0．000），其中以15μmol／L SV组NIM细胞变化最明显。15μmol／L SV联合ATRA处理NB4细胞培养72h CD11b（89．46％±9．13％）和hDMP1（626．9±56．9）表达水平明显高于ATRA（71．27％±7．27％和421．8±38．3）和SV（62．41％±6．37％和241．4±21．9）单药处理（均P〈0．05），两药合用具有明显交互作用（F=4．09，P=0．025和F=29．58，P=0．000）。联合用药对NB4细胞抑制率和Annexin—V表达水平差异无统计学意义。结论SV以剂量依赖方式体外抑制NB4细胞生长，诱导NB4细胞的分化，促进凋亡，降低WT1表达，升高hDMP1表达，提示SV具有协同治疗急性早幼粒细胞白血病的潜能。

Objective To investigate the effects of simvastatin （SV） plus all-trans retinoic acid （ATRA） on the proliferation, differentiation, apoptosis and WT1/hDMP1 gene expression profiles of human promyelocytie leukemia cell line NB4. Methods The NB4 cell was incubated with simvastatin and ATRA alone or in combination. And the NB4 cell without any treatment was adopted as a normal control. The ceils of different groups were collected at 24, 48 and 72 h post-incubation. Their morphological changes were observed after Wright staining. The method of MTT was employed to assay the growth inhibition rate and flow cytometry was used to detect the early-stage ratios of apoptosis and cell necrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction （RT-PCR） was used to detect the WT1/hDMP1 gene expression levels. Results The cell inhibition rates increased gradually （ F = 7.15, P = 0. 000） at 15, 10 and 5 μmol/L SV respectively. And so did the expression levels of CDllb （F = 3.41, P = 0. 014） and Annexin-V （ F = 43.38, P = 0. 000 ）. However the expression levels of WT1 decreased gradually （ F = 5. 35, P = 0. 001 ） reversely with the elevated levels of hDMP1 （ F = 22. 61, P = 0. 000 ）. Furthermore the NB4 cell exhibited the most significant changes at 15 μmol/L SV. After a 72-hour incubation, the expression levels of CD1 l b（ 89.46% ± 9. 13% ）and hDMP1 （626. 9 ± 56. 9）in NB4 cells at 15 μmol/L SV plus 0. 5 μmol/L ATRA were significantly higher than those with ATRA（71.27% ± 7.27% , P = 0. 000 and 421.8 ±38.3, P=0.003 in each）and SV alone（62.41% ±6.37%, P =0.003 and 241.4 ±21.9, P= 0. 003 in each）. A combination of 15 μmol/L SV with 0. 5 μmol/L ATRA displayed obvious interactions with the expressions of CD11 b and hDMP1 （ F = 4. 09, P = 0. 025 and F = 29. 58, P = 0. 000 in each）. And there was no significant interaction for cell inhibition rates and Annexin-V expression. Conclusion Simvastatin in vitro inhibits the proliferation of NB4 cell, induces its differentiation and promotes its apoptosis. And the lowered expression of WT1 has a dose-dependent correlation with the elevated expression of hDMP1. It indicates that simvastatin has the synergistic in vitro anti-promyeloeytic potency.