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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 investigate the effects of cholesterol synthesis inhibitor Lovastatin (LOV) on the proliferation, apoptosis and differentiation of NB4 leukemia cells. Methods NB4 leukemia cells as a model, MTT colorimetric method, trypan blue exclusion method first observed the effects of LOV on cell proliferation and viability. NB4 reduction assay, DNA gel electrophoresis, flow cytometry cell cycle analysis, TUNEL, RT PCR semi-quantitative determination of bcl 2 mRNA levels observed by LOV on NB4 induced differentiation and apoptosis in vitro. Finally, the expression levels of H, K, N ras genes were semi-quantitatively determined by RT-PCR and the membrane-bound p2 1 Ras protein was determined by flow cytometry to explore the mechanism of LOV. Results ①LOV inhibited the proliferation of NB4 cells with IC50 of 12.59μmol L. ② LOV induced apoptosis of NB4 cells, affecting the cell cycle progression, cell arrest in G1 S phase. LOV in the process of induction of apoptosis with the extension of time, bcl 2 expression decreased. ③ LOV does not affect NB4 cell differentiation. ④LOV effect on NB4 cells, H, K, N ras gene transcription level did not change, but the membrane surface p2 1Ras protein levels decreased over time. Conclusion LOV can inhibit the proliferation and induce the apoptosis of NB4 cells, and block the cell cycle progression in G1 phase. bcl 2 is involved in the gene regulation of LOV-induced NB4 cell apoptosis. LOV had no effect on NB4 cell differentiation. Suppression of p2 1 Ras protein prenylation and block of p2 1 Ras locating on the cell membrane may be the main mechanism by which LOV affects NB4 cells.