【作者】 赵菲；

【导师】 陈燕；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 目的：多发性骨髓瘤(multiple myeloma, MM)是一种起源于B淋巴细胞并能分泌大量单克隆免疫球蛋白的恶性浆细胞病,目前仍属一种难治性疾病。大量研究表明表观遗传学的失衡与多发性骨髓瘤的发生具有密切关系。本实验主要研究雷公藤内酯醇(triptolide, TPL)对多发性骨髓瘤细胞RPMI8226的细胞增殖,细胞周期阻滞,细胞凋亡,组蛋白H3、H4乙酰化,组蛋白H3K9、H3K27甲基化水平以及相应组蛋白去乙酰化酶、组蛋白甲基化酶的影响,探讨其抗肿瘤机制。方法：采用MTT比色法检测雷公藤内酯醇作用后多发性骨髓瘤RPMI8226细胞的增殖活性,并与外周血单个核细胞作比较,PI单染法流式细胞术检测细胞周期,Annexin V-FITC/PI双染法流式细胞术、TUNEL检测细胞凋亡,分光光度法检测Caspase-3和Caspase-9活性,Western blot免疫印迹技术检测组蛋白H3、H4乙酰化,组蛋白H3K9、H3K27甲基化水平以及HDAC8、SUV39H1和EZH2蛋白的表达,RT-PCR和Real-time RT-PCR技术分别检测hdac8和suv39h1、ezh2的基因水平,免疫荧光共聚焦观察HDAC8、SUV39H1和EZH2的亚细胞定位和表达。结果：雷公藤内酯醇能明显抑制RPMI8226细胞的增殖,其抑制作用呈时间、剂量依赖性,36h、48h和72h的的IC50值分别为(105.37±0.19)、(76.89±0.02)和(39.17±0.13)nmol/L;能引起RPMI8226细胞的G0/G1细胞周期阻滞；诱导RPMI8226细胞capsase-依赖性凋亡；能明显升高组蛋白H3和H4的乙酰化水平,降低组蛋白H3K9me3和H3K27me3的甲基化水平；经雷公藤内酯醇干预后,RPMI8226细胞HDAC8, SUV39H1和EZH2蛋白和mRNA水平均明显下降。结论：雷公藤内酯醇能够明显抑制RPMI8226的增殖,引起G0/G1细胞周期阻滞,并能诱导凋亡；通过降低RPMI8226细胞HDAC8的表达从而提高组蛋白H3和H4的乙酰化水平,通过抑制RPMI8226细胞SUV39H1和EZH2来降低组蛋白H3K9me3和H3K27me3的甲基化水平,这可能是雷公藤内酯醇抗多发性骨髓瘤作用的机制之一。目的：本实验主要研究雷公藤内酯醇对多发性骨髓瘤细胞U266的细胞增殖,细胞周期阻滞,细胞凋亡,组蛋白H3K4、H3K27和H3K36甲基化水平以及相应组蛋白甲基化酶的影响,探讨其抗肿瘤机制。方法：采用MTT比色法检测雷公藤内酯醇作用后多发性骨髓瘤U266细胞的增殖活性,PI单染法流式细胞术检测细胞周期,Annexin V-FITC/PI双染法流式细胞术,分光光度法检测Caspase-3和Caspase-9活性,Western blot免疫印迹技术检测组蛋白H3K4、H3K27和H3K36甲基化水平以及SMYD3.EZH2和NSD1蛋白的表达,RT-PCR分别检测smyd3、ezh2和nsd1的基因水平,免疫荧光共聚焦观察SMYD3、EZH2和NSD1的亚细胞定位和表达。结果：雷公藤内酯醇能明显抑制U266细胞的增殖,其抑制作用呈时间、剂量依赖性；能引起G2/M期细胞周期阻滞；诱导capsase-依赖性细胞凋亡；降低组蛋白H3K4me3、H3K27me3和H3K36me3的甲基化水平；经雷公藤内酯醇干预后,U266细胞SMYD3、EZH2和NSD1蛋白和mRNA水平均明显下降。结论：雷公藤内酯醇能够明显抑制U266细胞的增殖,引起G2/M期细胞周期阻滞并能诱导凋亡；通过降低U266细胞组蛋白甲基化酶SMYD3、EZH2和NSD1的表达从而降低组蛋白H3K9me3和H3K27me3的甲基化水平,这可能是雷公藤内酯醇抗多发性骨髓瘤作用的机制之一。目的：本实验主要研究雷公藤内酯醇对多发性骨髓瘤U266细胞的细胞凋亡及其通过调控RIZ1对组蛋白H3K9mel甲基化水平的影响,探讨其抗肿瘤机制。方法：Hoechst 33258染色法检测U266细胞凋亡,RT-PCR检测Caspase-3 mRNA表达,Western blot及流式细胞术检测RIZ1的蛋白表达,RT-PCR检测rizl的基因水平,Western blot检测组蛋白H3K9mel的蛋白表达,免疫荧光共聚焦观察H3K9mel的亚细胞定位及表达。结果：Hoechst 33258染色显示雷公藤内酯醇作用于U266细胞,可见典型的凋亡形态学改变如染色质浓缩、边缘化分割成块状,核膜裂解,出现凋亡小体。随着药物浓度的增加,凋亡细胞也随之数量增多；能上调U266细胞中RIZ1的蛋白和mRNA表达,并且具有明确的时效和量效关系；另外,RIZ1定位于U266细胞核内,雷公藤内酯醇作用后荧光强度显著升高。从而推测雷公藤内酯醇可能通过上调U266细胞RIZ1的表达,从而抑制U266细胞增殖和诱导凋亡。结论：雷公藤内酯醇抑制多发性骨髓瘤U266细胞增殖,诱导细胞发生caspase-依赖的凋亡。在U266细胞系中,RIZ1低表达,同时雷公藤内酯醇能够通过诱导RIZ1的表达上调组蛋白H3K9mel甲基化水平,这为探讨雷公藤内酯醇抗肿瘤机制提供一个新的视角。更多还原

【Abstract】 Objective:Multiple myeloma is a kind of malignant plasma disease that originated from B lymphocyte and secrete great amount of monoclonal immunoglobulin. It is still one of the refractory diseases at present. Numerous studies show that there is an intensive relationship between the disequilibrium of epigenetics and the occurance of multiple myeloma. Here we investigated the effects of triptolide on cell proliferation, cell cycle arrest, apoptosis, histone H3 and H4 acetylation, histone H3K9 and H3K27 trimethylation, and expression of the corresponding histone deacetylase and histone methyltransferases in vitro, trying to explore the anti-myeloma mechanism.Methods:The effect of triptolide on the growth of RPMI8226 was studied by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay and compared with PBMC. Effects of triptolide on the cell cycle distribution of RPMI8226 cells were studied by propidium iodide method. Apoptosis was detected by TUNEL and Annexin V-FITC/PI double-labeled cytometry and Caspase-3 and Caspase-9 activities were measured by spectrophotometry. The protein expression of acetyl-histone H3 and H4, histone H3K9me3, H3K27me3 and H3K36me3, HDAC8, SUV39H1 and EZH2 were determined by Western blot. The mRNA expression of hdac8 was assessed by RT-PCR, while the mRNA expression of suv39hl and ezh2 were measured by Real-time RT-PCR. Confocal microscopy was used to evaluate the subcellular disposition and expression of HDAC8, SUV39H1 and EZH2.Results:Triptolide inhibited the proliferation of RPMI8226 dose-and time-dependently, IC50for24h,48hand72h was 105.37±0.19,76.89±0.02 and39.17±0.13 nmol/L, respectively. Triptolide caused G0/G1 arrest and induced apoptosis in a time-and dose-dependent manner. Triptolide remarkably increased the acetylation of histone H3 and H4 by decreasing the expression of HDAC8 while declined histone H3K9me3 and H3K27me3 via inhibiting the expression of SUV39H1 and EZH2 respectively.Conclusion:Triptolide can inhibit cell proliferation, cause G0/G1 arrest and induce caspase dependent-apoptosis of RPMI8226 cells significantly. Triptolide remarkably increased the acetylation of histone H3 and H4 by decreasing the expression of HDAC8 while declined histone H3K9me3 and H3K27me3 via inhibiting the expression of SUV39H1 and EZH2 respectively, which is possibly the anti-myeloma mechanism of triptolide.Objective:To investigated the effect of triptolide on cell proliferation, cell cycle arrest, apoptosis, histone H3K4, H3K27 and H3K36 trimethylation, and the expression of corresponding histone methyltransferases in vitro, trying to explore the anti-myeloma mechanism.Methods:The effect of triptolide on the growth of U266 was studied by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium(MTT) assay and compared with PBMC. Effects of triptolide on the cell cycle distribution of U266 cells were studied by propidium iodide method. Apoptosis was detected by Annexin V-FITC/PI double-labeled cytometry and Caspase-3 and Caspase-9 activities were measured by spectrophotometry. The protein expression of histone H3K4me3, H3K27me3 and H3K36me3, SMYD3, EZH2 and NSD1 were determined by Western blot. The mRNA expression of smyd3, ezh2 and nsdl were assessed by RT-PCR. Confocal microscopy was used to evaluate the subcellular disposition and expression of SMYD3, EZH2 and NSD1.Results:Triptolide inhibited the proliferation of U266 cells significantly with the IC50 of 157.19±0.38,57.19±0.38 and 41.20±0.13 nmol/L for 24 h,48 h and 72 h respectively. Triptolide caused G2/M cell cycle arrest and caspase-dependent apoptosis. In U266 cells triptolide reduced histone H3K4me3, H3K27me3 and H3K36me3 along with the decline of the mRNA and protein expression of SMYD3,EZH2 and NSD1.Conclusion:Triptolide presented potent effects on growth inhibition, G2/M cell cycle arrest and induction of caspase-dependent apoptosis in U266 cells in vitro. The study provides the first evidence that triptolide induces epigenetic alterations by regulating histone lysine methylation, offering a novel view of anti-myeloma mechanism of triptolide.Objective:To investigate the effects of triptolide on the apoptosis in human multiple myeloma cell line U266 in vitro, and the regulation of histone H3K9 monomethylation (H3K9me1) via RIZ1 by triptolide.Methods:The effect of triptolide on cell apoptosis was detected through Hoechst 33258 staining and the mRNA expresson of caspase-3 was measured by RT-PCR. Western blot, flow cytometry and RT-PCR were applied to assess the expression of RIZ1, while the location and expression of H3K9mel regulated by RIZ1 were detected by confocal microscope and Western blot.Results:It was shown that triptolide could induce typical apoptotic morphological changes and concentration-dependent apoptosis that was caspase-3 dependent. Compared with PBMC from healthy donors, the protein expression of RIZ1 in U266 cells was relatively low, while the mRNA and protein expression of RIZ1 were strikingly increased by triptolide in a concentration-dependent manner. The protein expression of histone H3K9me1 regulated via RIZ1 was detected significantly elevated by triptolide. Conclusion:Triptolide exerted caspase-dependent apoptosis-inducing potency in U266 cells. The expression of RIZ1 in U266 cells was relatively low while it appeared remarkably upregulatd when treated with triptolide.更多还原