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
Objective： To investigate the effects of small hairpin RNA（shRNA） targeting Bcl-2 on the growth of NB4 cell line. Methods： Two of pairs oligonucleotides for short hairpin expression targeting the coding region of ...Objective： To investigate the effects of small hairpin RNA（shRNA） targeting Bcl-2 on the growth of NB4 cell line. Methods： Two of pairs oligonucleotides for short hairpin expression targeting the coding region of Bcl-2 mRNA were designed and chemically synthesized. Annealed oligonucleotides were inserted into Pgenesil-1 vector downstream of U6 promoter to construct RNAi plasmid. Oligonucleotide with a scrambled sequence was used as a negative control. Recombinant expression vector was identified by enzyme cutting and sequencing. Bcl-2 shRNAs were transfected into NB4 cell with Lipofectamine 2000. Western-Blot of Bcl-2 protein expression in NB4 cells was performed after transfection. The inhibition of cell growth was assessed by a MTT assay. Results： Enzyme cutting and sequencing showed that the insertion sequence was correct. Western-Blot assay showed that the expression level of Bcl-2 protein in NB4 cells decreased after Bcl-2 shRNAs treatment. There was no difference in Bcl-2 protein levels between control shRNA and untreated cells. Viable cells at 72 and 96 h after treatment with Bcl-2 shRNAs were less than that after treatment with control shRNAs and untreated NB4 cells, respectively （P〈0.05）. Control shRNA had no significant effect on the growth of the cells. Conclusion： Bcl-2 shRNA could effectively inhibit the growth of NB4 cells. Bcl-2 shRNA might be an effective anti-leukemia candidate.展开更多
Objective: To investigate the effects of curcumin on the proliferation of NB4 cells and the acetylation of histone H3 and no-histone p53. Methods: NB4 cells were cultured and treated with or without curcumin at differ...Objective: To investigate the effects of curcumin on the proliferation of NB4 cells and the acetylation of histone H3 and no-histone p53. Methods: NB4 cells were cultured and treated with or without curcumin at different concentration (50 μ mol/L, 25μ mol/L, 12.5μ mol/L, 6.25μ mol/L, 3.125μ mol/L) at various time points (oh, 4h, 8h, 12h, 24h). Western blot analysis was performed to determine the level of acetylated histone H3, p53 and acetylated p53, MTT assay was performed to examine the proliferation of NB4 cells. Results: the proliferation of NB4 cells was inhibited by curcumin in a time- and dose-dependent manner, the IC50 at 24h and 36h were 40μ mol/L and 25μ mol/L respectively. The levels of acetylated histone H3 and acetylated p53 were increased obviously. Conclusion: curcumin possessed the founction of deacetylases inhibitor, increased the level of acetylated histone H3 and the expression of tumor suppressor p53, enhanced later’s activity, and inhibited the proliferation of NB4 cell.展开更多
Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and...Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR Ⅰ adapters. Then the cDNAs were digested with Xho Ⅰ, and less than 400 bp cDNA fragment was removed by Sephacryl S400 spin column, the remaining were ligated with λZAP vector. The recombinants were packaged in vitro , and a small portion of packaged phage was used to infect E. coli XL1 Blue MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK CMV phagemid was digested by Xho Ⅰ and EcoR Ⅰ. The results showed that the NB4 cell line cDNA library consisting of 1.65×10 6 recombinant bacteriophages was constructed with the recombinant ratio of 99.6 %. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.展开更多
Objective： To investigate the effects of staphyococcal enterotoxin A （SEA） on the cytotoxicity of T cells stimulated by PML-RARa peptide in vitro. Methods： Peripheral blood mononuclear cells （MNC） from healthy d...Objective： To investigate the effects of staphyococcal enterotoxin A （SEA） on the cytotoxicity of T cells stimulated by PML-RARa peptide in vitro. Methods： Peripheral blood mononuclear cells （MNC） from healthy donor were obtained by density gradient centrifugation on Ficoll-Hypaque, MNC were cultured with PML-RARa peptide and SEA for 20 days. After induction, the cytotoxicity of T cells induced against NB4 and K562 cell lines were examined by Cell Counting Kit-8 （CCK-8）. The CD4 and CD8 surface markers on the harvested CD3^＋ T cells were detected by flow cytometry （FCM）. Results： The cytotoxicity of T cells induced by PML-RARa peptide with SEA was higher than that of T cells induced only by PML-RARa peptide against NB4 cells. The FCM assay showed that the ratio of CD4^＋/CD8^＋ T cells were gradually decreased in both groups of PML-RARα peptide whether with SEA or not at the intervals of day 5,10 and 20 day after induction, but the most significantly decreased by PML-RARe peptide with SEA. Conclusion： The specific cytotoxicity of T cells induced by PML-RARa peptide against NB4 cells could be enhanced with superantigen SEA.展开更多
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
本研究探讨葛根有效成分对恶性白血病可能的凋亡诱导作用及其分子机制。采用葛根总黄酮(flavonoids of puerarin,PR)处理人早幼粒细胞白血病(APL)细胞株NB4,用MTT法检测细胞增殖抑制率;FITC-Annexin V/PI双染法检测细胞凋亡率;实时定量...本研究探讨葛根有效成分对恶性白血病可能的凋亡诱导作用及其分子机制。采用葛根总黄酮(flavonoids of puerarin,PR)处理人早幼粒细胞白血病(APL)细胞株NB4,用MTT法检测细胞增殖抑制率;FITC-Annexin V/PI双染法检测细胞凋亡率;实时定量PCR检测pml/rarα、bcl-2、survivin基因表达;Western blot检测JNK、p38MAPK、FasL及caspase相关酶的变化。结果发现,PR能明显抑制NB4细胞增殖,并诱导细胞凋亡。随着PR浓度的增加,pml/rarα、bcl-2及survivin基因在mRNA水平表达下调,JNK、FasL、caspase3及caspase8蛋白表达增加,与PR浓度呈正相关;PR联合三氧化二砷(arsenic trioxide,ATO)处理后上述作用更为明显。结论:PR诱导NB4细胞凋亡的机制可能与JNK相关信号分子的活化有关;PR联合ATO具有协同诱导NB4细胞凋亡的作用。展开更多
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.
基金This work was supported by a grant from the Natural Science Program Foundation of Guangdong Province (No.04010446)
文摘Objective： To investigate the effects of small hairpin RNA（shRNA） targeting Bcl-2 on the growth of NB4 cell line. Methods： Two of pairs oligonucleotides for short hairpin expression targeting the coding region of Bcl-2 mRNA were designed and chemically synthesized. Annealed oligonucleotides were inserted into Pgenesil-1 vector downstream of U6 promoter to construct RNAi plasmid. Oligonucleotide with a scrambled sequence was used as a negative control. Recombinant expression vector was identified by enzyme cutting and sequencing. Bcl-2 shRNAs were transfected into NB4 cell with Lipofectamine 2000. Western-Blot of Bcl-2 protein expression in NB4 cells was performed after transfection. The inhibition of cell growth was assessed by a MTT assay. Results： Enzyme cutting and sequencing showed that the insertion sequence was correct. Western-Blot assay showed that the expression level of Bcl-2 protein in NB4 cells decreased after Bcl-2 shRNAs treatment. There was no difference in Bcl-2 protein levels between control shRNA and untreated cells. Viable cells at 72 and 96 h after treatment with Bcl-2 shRNAs were less than that after treatment with control shRNAs and untreated NB4 cells, respectively （P〈0.05）. Control shRNA had no significant effect on the growth of the cells. Conclusion： Bcl-2 shRNA could effectively inhibit the growth of NB4 cells. Bcl-2 shRNA might be an effective anti-leukemia candidate.
基金this work was supported by NationNatural Science Foundation of China(No. 30271672).
文摘Objective: To investigate the effects of curcumin on the proliferation of NB4 cells and the acetylation of histone H3 and no-histone p53. Methods: NB4 cells were cultured and treated with or without curcumin at different concentration (50 μ mol/L, 25μ mol/L, 12.5μ mol/L, 6.25μ mol/L, 3.125μ mol/L) at various time points (oh, 4h, 8h, 12h, 24h). Western blot analysis was performed to determine the level of acetylated histone H3, p53 and acetylated p53, MTT assay was performed to examine the proliferation of NB4 cells. Results: the proliferation of NB4 cells was inhibited by curcumin in a time- and dose-dependent manner, the IC50 at 24h and 36h were 40μ mol/L and 25μ mol/L respectively. The levels of acetylated histone H3 and acetylated p53 were increased obviously. Conclusion: curcumin possessed the founction of deacetylases inhibitor, increased the level of acetylated histone H3 and the expression of tumor suppressor p53, enhanced later’s activity, and inhibited the proliferation of NB4 cell.
文摘Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR Ⅰ adapters. Then the cDNAs were digested with Xho Ⅰ, and less than 400 bp cDNA fragment was removed by Sephacryl S400 spin column, the remaining were ligated with λZAP vector. The recombinants were packaged in vitro , and a small portion of packaged phage was used to infect E. coli XL1 Blue MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK CMV phagemid was digested by Xho Ⅰ and EcoR Ⅰ. The results showed that the NB4 cell line cDNA library consisting of 1.65×10 6 recombinant bacteriophages was constructed with the recombinant ratio of 99.6 %. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.
基金Supported by grants from the Science and Technology Commission of Guangdong Province (No.06025169,No.2005B50301016)the Key Laboratory of Pathophysiology of Jinan University
文摘Objective： To investigate the effects of staphyococcal enterotoxin A （SEA） on the cytotoxicity of T cells stimulated by PML-RARa peptide in vitro. Methods： Peripheral blood mononuclear cells （MNC） from healthy donor were obtained by density gradient centrifugation on Ficoll-Hypaque, MNC were cultured with PML-RARa peptide and SEA for 20 days. After induction, the cytotoxicity of T cells induced against NB4 and K562 cell lines were examined by Cell Counting Kit-8 （CCK-8）. The CD4 and CD8 surface markers on the harvested CD3^＋ T cells were detected by flow cytometry （FCM）. Results： The cytotoxicity of T cells induced by PML-RARa peptide with SEA was higher than that of T cells induced only by PML-RARa peptide against NB4 cells. The FCM assay showed that the ratio of CD4^＋/CD8^＋ T cells were gradually decreased in both groups of PML-RARα peptide whether with SEA or not at the intervals of day 5,10 and 20 day after induction, but the most significantly decreased by PML-RARe peptide with SEA. Conclusion： The specific cytotoxicity of T cells induced by PML-RARa peptide against NB4 cells could be enhanced with superantigen SEA.
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.
