辛伐他汀联合全反式维甲酸诱导NB4细胞分化凋亡过程中apoM基因表达的变化

apo M gene expression profiles during the process of simvastatin with all transretinoic acid inducing NB4 differentiation and apoptosis

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NB4细胞增殖、分化与凋亡的影响,以及载脂蛋白M(apo M)基因的表达变化。方法以不同浓度辛伐他汀联合ATRA处理NB4细胞,取对数生长期各组细胞分别进行细胞形态观察;MTT法观察细胞增殖能力;流式细胞测定NB4细胞分化指标CD11b和细胞凋亡指标AnnexinⅤ/propidium iodide变化;Real-time RT-PCR测定apoM基因表达。结果重复三次实验结果显示:15μmol/L SV,10μmol/L SV和5μmol/L SV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),AnnexinV表达水平逐渐增高(F=43.38,P=0.000),apo M基因表达水平逐渐增高(F=50.31,P=0.000),其中以15μmol/L SV组NB4细胞变化最明显。辛伐他汀和ATRA两药合用CD11b表达协同升高,具有明显交互作用(F=4.09,P=0.025)。ATRA处理NB4细胞后其apo M基因表达水平逐渐下降(F=46.05,P=0.000),而辛伐他汀联合ATRA处理NB4细胞后培养不同时间,apo M基因表达水平不如辛伐他汀和ATRA单药处理变化明显,15μmol/L SV联合ATRA处理NB4细胞72hapo M基因表达水平(32.87±3.60)较24h(24.73±1.78)升高(P<0.05),提示ATRA可以拮抗辛伐他汀引起NB4细胞apo M基因表达水平代偿性升高。结论辛伐他汀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,升高apo M基因表达,而ATRA可以拮抗辛伐他汀引起apo M代偿性升高,两药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 transretinoic acid（ATRA） on the proliferation,differentiation,apoptosis of human promyelocytic leukemia cell line NB4 and apo M gene expression profiles.Methods The NB4 cells were incubated with Simvastatin plus all transretinoic acid（ATRA）.The morphological changes of cell groups in exponential phase were observed after Wright stain.The method of MIT was used to assay the growth proliferation rate and flow cytometry was used to detect changes of the NB4 differentiation index CD11b and the apoptosis index AnnexinⅤ/propidium.Real time quantitative reverse transcriptase polymerase chain reaction（RT PCR） was used to detect the apo M gene expression levels.Results Treated with 15 μmol/L SV,10 μmol/L SV and 5 μmol/L SV respectively in three independent tests,with the NB4-cells growth,the cell inhibition rates gradually increased（F=7.15,P=0.000）,so did the CD11b expression levels（F=3.41,P=0.014）,AnnexinV expression levels（F=43.38,P=0.000） and apo M gene expression levels（F=5.31,P=0.000）.And the NB4 cells treated with 15 μmol/L SV exhibited the most significant changes.Treated with ATRA,the apo M gene expression levels gradually decreased with the NB4-cells growth（F=46.05,P=0.000）.Combination of simvastatin and ATRA displayed obvious interaction for CD11b expression（F=4.09,P=0.025）,yet no interaction for the growth inhibition rate,AnnexinV and apo M expression levels.The apo M gene expression levels of NB4 cells treated with 15 μmol/L SV combined with 0.5 μmol/L ATRA after 72 h incubation was mildly elevated than that after 24 h incubation（32.87±3.60 vs 24.73±1.78,P0.05）,indicating that ATRA might antagonize the adaptive defensive apo M gene expression elevation induced by simvastatin.Conclusion Simvastatin in vitro inhibits NB4-cell proliferation,induces cell differentiation and promotes cell apoptosis as well as increases the apo M expression dose dependently.However,ATRA might antagonize the adaptive defensive apo M gene expression elevation induced by simvastatin,indicating that simvastatin plus ATRA has the synergistic anti-promyelocytic potency in vitro.