Objective: To investigate the effects of anti-PML (promyelocytic leukemia) or anti-PML/RAR( (promyelocytic leukemia/retionic acid receptor() antisense oligonucleotides on cell growth, expression of PML-RAR( mRNA and P...Objective: To investigate the effects of anti-PML (promyelocytic leukemia) or anti-PML/RAR( (promyelocytic leukemia/retionic acid receptor() antisense oligonucleotides on cell growth, expression of PML-RAR( mRNA and PML-RAR(/PML protein location of NB4 cell lines. Methods: RT-PCR was used for detecting PML-RAR( mRNA expression, trypan blue exclusion for cell count, methylcellose assay for leukemic colony forming unit detection, immuno- fluorescence for PML-RAR(/PML protein location. Results: Both anti-PML start codon region antisence (STAS) and anti-PML-RAR( fusion region antisence (FUAS) could inhibit cell growth and the formation of acute myelocytic colony forming unit of cells(AML-CFU). Cells become partial differentiated at days 5, being more obvious in FUAS-treated cells than in STAS ones. Down regulation of PML-RAR( mRNA expression occurred at 24 hours in STAS and FUAS-treated cells and maintained for up to 72 hours. Immuno-fluorescence analysis with anti-PML monoclonal antibody showed a remarkable decrease even complete disappearance of microgranules. The residual granules became enlarged as discrete dots (<10 per cell), similar to normal POD structure in some STAS-treated cells at 24 hours. At 72 hours, nearly all the granules disappeared. Similar changes were observed in FUAS-treated cells. Conclusion: Both PML and PML-RAR( antisence oligonucleotides can specially block the expression of PML-RAR( at mRNA and protein levels. PML protein is implicated in the regulations of cell differentiation.展开更多
Objective: To evaluate whether realgar could down-regulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in acute promyelocytic leukemia cell line-NB4 cells. Methods: The expre...Objective: To evaluate whether realgar could down-regulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in acute promyelocytic leukemia cell line-NB4 cells. Methods: The expression of hTERT-mRNA was analyzed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Flow cytometry using PI staining was applied to analyze the cell cycle and apoptosis. Results: Treatment of NB4 cells with 155, 300, 600 μg/L realgar reduced telomerase activity significantly accompanying with decrease of hTERT-mRNA and increasing cell apoptosis. G2/M phase arrest appeared when treated with realgar in 300, 600 μg/L. Conclusion: It is suggested that telomerase activity of NB4 cells can be specifically inhibited by realgar through the down-regulation of hTERT gene expression. G2/M phase arrest and apoptosis by realgar in NB4 cells might be related to the reduction of telomerase activity and hTERT-mRNA expression.展开更多
目的:探讨小檗胺对白血病细胞系NB4生长的影响及其机制。方法:体外培养人白血病细胞株NB4细胞,经不同浓度小檗胺处理不同时间后,MTT法检测药物对NB4细胞的抑制作用;细胞形态学观察和DNA琼脂糖电泳检测细胞凋亡;流式细胞仪检测DNA含量及...目的:探讨小檗胺对白血病细胞系NB4生长的影响及其机制。方法:体外培养人白血病细胞株NB4细胞,经不同浓度小檗胺处理不同时间后,MTT法检测药物对NB4细胞的抑制作用;细胞形态学观察和DNA琼脂糖电泳检测细胞凋亡;流式细胞仪检测DNA含量及细胞周期;巢式PCR及RT-PCR检测PM L/RARα融合基因及Surv iv in基因表达及流式细胞仪检测Caspase3阳性表达率。结果:经不同浓度小檗胺处理不同时间后,NB4细胞生长出现明显的抑制,并呈现明显的时间剂量依赖性,48 h IC50为3.860μg/m l;细胞形态学观察可见细胞凋亡现象,DNA琼脂糖电泳显示典型的凋亡梯形图;流式细胞仪检测发现细胞凋亡率明显增加,经48 h作用后,凋亡率从2.83%增至12μg/m l时的58.44%;虽未发现药物作用后PM L/RARα基因表达量的改变,但检测到NB4细胞经药物作用后Surv iv in基因表达量的减少和Caspase3表达的增加,Caspase3阳性率从对照组的2.06%增至12μg/m l时的70.89%。经相关性分析,随着细胞Surv iv in表达水平的下降和Caspase3表达水平的增高,NB4细胞凋亡率相应地增高。结论:小檗胺对NB4细胞有增殖抑制作用,诱导细胞凋亡可能是其抗肿瘤作用的重要机制之一,但不是通过作用于NB4细胞特异性融合基因PM L/RARα发挥作用的,其抗凋亡的可能分子机制之一是抑制Surv iv in基因的表达和增加Caspase3的表达。展开更多
目的观察丹参酮ⅡA(Tanshinone,TanⅡA)联合三氧化二砷(ATO)对人急性早幼粒细胞白血病细胞株(NB4细胞)凋亡的作用;并通过观察两药联合诱导NB4细胞凋亡时p53、Bcl-2表达的变化,探讨其可能的机制。方法通过Annexin V FITC/PI荧光染色观察...目的观察丹参酮ⅡA(Tanshinone,TanⅡA)联合三氧化二砷(ATO)对人急性早幼粒细胞白血病细胞株(NB4细胞)凋亡的作用;并通过观察两药联合诱导NB4细胞凋亡时p53、Bcl-2表达的变化,探讨其可能的机制。方法通过Annexin V FITC/PI荧光染色观察1.0μg·mL-1TanⅡA分别联合0.25、0.5、1.0μmol·L-1的ATO作用于NB4细胞的形态学变化;流式细胞术Annexin V-FITC/PI法测定各组细胞凋亡率;流式细胞仪检测各组细胞p53、Bcl-2的表达。结果 (1)1.0μg·mL-1TanⅡA分别与0.5、1.0μmol·L-1的ATO联合作用NB4细胞24、48和72h的凋亡率均较相应单独用药组凋亡率明显增高(P<0.05);染色荧光显微镜观察l.0μg·mL-1TanⅡA联合1.0μmol·L-1ATO实验组较1.0μmol·L-1ATO对照组72h时更易见到中晚期凋亡细胞和死亡细胞;(2)1.0μg·mL-1TanⅡA与0.5、1.0μmol·L-1的ATO联合作用NB4细胞48h、72h表达p53蛋白的细胞较1.0μmol·L-1ATO单药组明显增多(P<0.05),作用高峰时间为72h;1.0μg·mL-1TanⅡA分别与0.25、0.5、1.0μmol·L-1的ATO联合作用NB4细胞24h时,表达Bcl-2蛋白的细胞数较1.0μmol·L-1ATO单药组显著下降(P<0.05),且与ATO的浓度呈负相关(r=-0.858,P=0.000)。结论联合1.0μg·mL-1TanⅡA可增强0.5、1.0μmol·L-1ATO诱导NB4细胞凋亡;TanⅡA联合ATO诱导NB4细胞p53表达增强和Bcl-2表达减少可能是其促凋亡机制之一。展开更多
基金the National Natural Science Foundation of China(No. 39590291).
文摘Objective: To investigate the effects of anti-PML (promyelocytic leukemia) or anti-PML/RAR( (promyelocytic leukemia/retionic acid receptor() antisense oligonucleotides on cell growth, expression of PML-RAR( mRNA and PML-RAR(/PML protein location of NB4 cell lines. Methods: RT-PCR was used for detecting PML-RAR( mRNA expression, trypan blue exclusion for cell count, methylcellose assay for leukemic colony forming unit detection, immuno- fluorescence for PML-RAR(/PML protein location. Results: Both anti-PML start codon region antisence (STAS) and anti-PML-RAR( fusion region antisence (FUAS) could inhibit cell growth and the formation of acute myelocytic colony forming unit of cells(AML-CFU). Cells become partial differentiated at days 5, being more obvious in FUAS-treated cells than in STAS ones. Down regulation of PML-RAR( mRNA expression occurred at 24 hours in STAS and FUAS-treated cells and maintained for up to 72 hours. Immuno-fluorescence analysis with anti-PML monoclonal antibody showed a remarkable decrease even complete disappearance of microgranules. The residual granules became enlarged as discrete dots (<10 per cell), similar to normal POD structure in some STAS-treated cells at 24 hours. At 72 hours, nearly all the granules disappeared. Similar changes were observed in FUAS-treated cells. Conclusion: Both PML and PML-RAR( antisence oligonucleotides can specially block the expression of PML-RAR( at mRNA and protein levels. PML protein is implicated in the regulations of cell differentiation.
基金Supported by Xi'an Foundation of Science and Technology Program(200016)
文摘Objective: To evaluate whether realgar could down-regulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in acute promyelocytic leukemia cell line-NB4 cells. Methods: The expression of hTERT-mRNA was analyzed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Flow cytometry using PI staining was applied to analyze the cell cycle and apoptosis. Results: Treatment of NB4 cells with 155, 300, 600 μg/L realgar reduced telomerase activity significantly accompanying with decrease of hTERT-mRNA and increasing cell apoptosis. G2/M phase arrest appeared when treated with realgar in 300, 600 μg/L. Conclusion: It is suggested that telomerase activity of NB4 cells can be specifically inhibited by realgar through the down-regulation of hTERT gene expression. G2/M phase arrest and apoptosis by realgar in NB4 cells might be related to the reduction of telomerase activity and hTERT-mRNA expression.
文摘目的:探讨小檗胺对白血病细胞系NB4生长的影响及其机制。方法:体外培养人白血病细胞株NB4细胞,经不同浓度小檗胺处理不同时间后,MTT法检测药物对NB4细胞的抑制作用;细胞形态学观察和DNA琼脂糖电泳检测细胞凋亡;流式细胞仪检测DNA含量及细胞周期;巢式PCR及RT-PCR检测PM L/RARα融合基因及Surv iv in基因表达及流式细胞仪检测Caspase3阳性表达率。结果:经不同浓度小檗胺处理不同时间后,NB4细胞生长出现明显的抑制,并呈现明显的时间剂量依赖性,48 h IC50为3.860μg/m l;细胞形态学观察可见细胞凋亡现象,DNA琼脂糖电泳显示典型的凋亡梯形图;流式细胞仪检测发现细胞凋亡率明显增加,经48 h作用后,凋亡率从2.83%增至12μg/m l时的58.44%;虽未发现药物作用后PM L/RARα基因表达量的改变,但检测到NB4细胞经药物作用后Surv iv in基因表达量的减少和Caspase3表达的增加,Caspase3阳性率从对照组的2.06%增至12μg/m l时的70.89%。经相关性分析,随着细胞Surv iv in表达水平的下降和Caspase3表达水平的增高,NB4细胞凋亡率相应地增高。结论:小檗胺对NB4细胞有增殖抑制作用,诱导细胞凋亡可能是其抗肿瘤作用的重要机制之一,但不是通过作用于NB4细胞特异性融合基因PM L/RARα发挥作用的,其抗凋亡的可能分子机制之一是抑制Surv iv in基因的表达和增加Caspase3的表达。
文摘目的观察丹参酮ⅡA(Tanshinone,TanⅡA)联合三氧化二砷(ATO)对人急性早幼粒细胞白血病细胞株(NB4细胞)凋亡的作用;并通过观察两药联合诱导NB4细胞凋亡时p53、Bcl-2表达的变化,探讨其可能的机制。方法通过Annexin V FITC/PI荧光染色观察1.0μg·mL-1TanⅡA分别联合0.25、0.5、1.0μmol·L-1的ATO作用于NB4细胞的形态学变化;流式细胞术Annexin V-FITC/PI法测定各组细胞凋亡率;流式细胞仪检测各组细胞p53、Bcl-2的表达。结果 (1)1.0μg·mL-1TanⅡA分别与0.5、1.0μmol·L-1的ATO联合作用NB4细胞24、48和72h的凋亡率均较相应单独用药组凋亡率明显增高(P<0.05);染色荧光显微镜观察l.0μg·mL-1TanⅡA联合1.0μmol·L-1ATO实验组较1.0μmol·L-1ATO对照组72h时更易见到中晚期凋亡细胞和死亡细胞;(2)1.0μg·mL-1TanⅡA与0.5、1.0μmol·L-1的ATO联合作用NB4细胞48h、72h表达p53蛋白的细胞较1.0μmol·L-1ATO单药组明显增多(P<0.05),作用高峰时间为72h;1.0μg·mL-1TanⅡA分别与0.25、0.5、1.0μmol·L-1的ATO联合作用NB4细胞24h时,表达Bcl-2蛋白的细胞数较1.0μmol·L-1ATO单药组显著下降(P<0.05),且与ATO的浓度呈负相关(r=-0.858,P=0.000)。结论联合1.0μg·mL-1TanⅡA可增强0.5、1.0μmol·L-1ATO诱导NB4细胞凋亡;TanⅡA联合ATO诱导NB4细胞p53表达增强和Bcl-2表达减少可能是其促凋亡机制之一。
