胆固醇合成抑制剂洛伐他汀对NB4细胞体外作用的研究

In vitro effect of lovastatin on NB4 promyelocytic leukemia cells

目的 研究胆固醇合成抑制剂洛伐他汀 (Lovastatin ,LOV)对NB4白血病细胞增殖、凋亡、分化的影响。方法 以NB4白血病细胞为模型 ,用MTT比色法、锥虫蓝拒染法首先观察LOV对细胞增殖及活力的影响。通过细胞形态学观察、NBT还原试验、DNA凝胶电泳、流式细胞术细胞周期分析、TUNEL、RT PCR半定量测定bcl 2mRNA水平 ,系统观察LOV对NB4体外诱导分化和凋亡的情况。最后利用RT PCR半定量测定H、K、N ras基因表达水平 ,结合流式细胞术进行胞膜p2 1Ras蛋白测定 ,探讨LOV作用机制。结果 ①LOV抑制NB4细胞增殖 ,IC50 为 12 .5 9μmol L。②LOV诱导NB4细胞凋亡 ,影响细胞周期进程 ,细胞阻滞于G1 S期。LOV在诱导细胞凋亡过程中随作用时间延长 ,bcl 2表达水平逐渐下降。③LOV不影响NB4细胞分化。④LOV作用于NB4细胞 ,H、K、N ras基因转录水平无变化 ,但膜表面p2 1Ras蛋白水平随时间进行性下降。结论 LOV抑制NB4细胞增殖并诱导凋亡 ,使细胞周期进程阻滞于G1 S期。bcl 2参与了LOV诱导NB4细胞凋亡的基因调控。LOV对NB4细胞分化无影响。p2 1Ras蛋白异戊二烯化受抑而阻滞p2 1Ras蛋白定位于细胞膜上可能是LOV影响NB4细胞的主要机制。

Objective To explore the effect of lovastatin(LOV) on cell growth,apoptosis and differen tiation of NB4 cells and elucidate some of the possible mechanism. Methods Cell proliferation and viability were analyzed by MTT assay and trypan blue exclusion assay, cell differentiation by NBT reduction.Cell morphological examination,DNA electrophoresis,flow cytometry,TUNEL in situ hybridization an d semi-quantitative RT-PCR were performed to determine cell apoptosis. H?K ?N-ras gene expression was investigated by RT-PCR and p21^(Ras) protein ex pression by flow cytometry. Results ①LOV inhibited the prolif eration of NB4 cells with a IC_(50) of 12.59 μmol/L. ②LOV induc ed apoptosis of NB4 cells,affected the cell cycle progression and arrested NB4 cells in G_1/S phase. Bcl-2 mRNA was down-regulated by LOV in a time-depende nt manner. ③LOV could not induce the differentiation of NB4. ④LOV did not incr ease the expression of H?K?N-ras mRNA, but decrease p21^(Ras) protein expr ession on the cellular membrane. Conclusion LOV can inhibit prolife ration,induce apoptosis and interfere with cell cycle progression,but not indu ce differentiation of NB4. Inhibition of p21^(Ras) protein isoprenylation is considered to be the major mechanism of LOV affecting NB4 cells.