地西他滨抗套细胞淋巴瘤胞株Jeko-1作用机制的研究

摘要

背景与目的:套细胞淋巴瘤(mantle cell lymphoma,MCL)是一种难治性的恶性肿瘤,预后差.地西他滨对骨髓异常增生综合征及难治性急性髓性白血病的治疗临床上已达成共识,但是目前鲜有地西他滨对MCL作用的报道.本文探讨地西他滨抗MCL细胞的作用机制.方法:以MCL细胞株Jeko-1作为研究对象,采用MTT法检测地西他滨对细胞增殖的影响,Annexin V/PI双重染色法检测其对细胞凋亡的影响,采用DiOC6(3)染色/流式细胞术检测Jeko-1细胞线粒体跨膜电位的丢失,Western blot检测细胞周期蛋白D1(Cyclin D1)及凋亡途径相关蛋白的表达,用亚硫酸氢盐测序(bisulfate sequencing PCR,BSP)技术检测地西他滨作用Jeko-1细胞前后PCDH8基因启动子CpG岛的甲基化水平变化.结果:地西他滨能有效抑制Jeko-1细胞的增殖并诱导其凋亡,凋亡率呈时间和剂量依赖性增加,并引起MCL细胞线粒体跨膜电位丢失,Cyclin D1蛋白表达水平降低,caspase 3、caspase 9表达水平增加.本研究另发现Jeko-1细胞中PCDH8基因启动子CpG岛的甲基化率为76.67%,经过0.5 μmol/L低浓度的地西他滨处理72 h后,其甲基化率下降至48.33%.结论:地西他滨在对MCL细胞的作用中,高浓度时起到细胞毒作用,低浓度时则具有去甲基化作用,为今后的临床治疗提供了新的理论依据.%Background and purpose: Mantle cell lymphoma (MCL) is one of the refractory malignant tumors with very poor prognosis.The treatment of decitabine against myelodysplastic syndrome and refractory acute myeloid leukemia has been widely applied.However, the effectiveness of decitabine against MCL cell lines has not been reported.This study was to explore the mechanisms of action of decitabine (DAC) against Jeko-1 MCL cell line.Methods: Jeko-1 cells were treated with different concentrations of DAC, cell growth rate was detected by MTT assay.Apoptosis of Jeko-1 cells treated with DAC was detected by using flow cytometry (FCM) after double staining with Annexin V-FITC/PI.The collapse of mitochondrial membrane potential of Jeko-1 cells treated with DAC was detected by FCM after staining with DiOC6(3).The expression of Cyclin D1 and apoptosis related proteins was detected by using Western blot.The CpG methylation of PCDH8 in Jeko-1 cells was detected by Bisulfite sequencing PCR (BSP).Results: Decitabine was capable of inhibiting cell growth, inducing apoptosis of MCL cells and disrupting mitochondrial membrane potential.DAC could not only decrease Cyclin D1 expression, but also increase expression of Caspase-3 and Caspase-9.The hypermethylation of PCDH8 gene (76.67％) was identified in Jeko-1 cells.After treatment with 0.5 μmol/L DAC for 72 h, the methylation of CpG on gene PCDH8 decreased to 48.33％.Conclusion: The mechanisms of decitabine against Jeko-1 MCL cell line was cytotoxic effect at high concentration and hypomethylation effect at low concentration.Therefore, this study provides a theory basis of new drug application for the treatment of MCL.