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OBJECTIVE@#To explore the effect of dichloromethane extraction phase of ethanol extract from stem of Patrinia scabiosaefolia Fisch.(DPSS) on proliferation and differentiation of K562 cells and its related mechanism.@*METHODS@#MTT assay was used to detect the effects of DPSS at 0, 25, 50, 100 and 200 μg/ml on the proliferation of K562 cells at 24, 48 and 72 hours. Flow cytometry was used to analyze the changes of cell cycle and apoptosis at 24 and 48 hours. Wright-Giemsa staining was used to observe the morphological changes of K562 cells. The cell surface antigens CD33 and CD11b were detected by flow cytometry.@*RESULTS@#The proliferation of K562 cells treated with different concentrations of DPSS was inhibited in a time-dose dependent manner (r=-0.96). Cell cycle analysis showed that with the increase of DPSS concentration, cells in G2/M phase increased (r=0.88), and cells were blocked in G2/M phase. Flow cytometry results showed that with the apoptosis rate of K562 cells was the highest when treated with 200 μg/ml DPSS for 48 h. Morphological observation showed that the K562 cell body increased, the amount of cytoplasm increased, the ratio of nucleus to cytoplasm decreased, and the nuclear chromatin was rough after DPSS treatment. Cell differentiation antigen, CD33 and CD11b, were positively expressed after treated with DPSS.@*CONCLUSION@#DPSS can induce apoptosis through cell cycle arrest, inhibit the proliferation of K562 cells, and induce K562 cells to differentiate into monocytes, which has a potential anti-leukemia effect.
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Humans , K562 Cells , Patrinia , Methylene Chloride/pharmacology , Apoptosis , Cell Proliferation , Cell DifferentiationABSTRACT
Natural killer (NK) cells are among the first in defense of the innate immune system by eliminating a variety of abnormal or stressed cells such as cancer cells or virus-infected cells. Individuals who exhibit low cytolytic NK cell activity are believed to be at higher risk of viral infection, tumorigenesis, and various other diseases of the immune system. Therefore, restoration of impaired NK cell function might be an essential step in immunostimulatory therapy of immunocompromised patients. Bacillus firmus is a non-pathogenic gram-positive bacterium of the environment, which possesses various immunomodulatory properties in vitro and in vivo. This retrospective study reports on the effect of B. firmus on the activity of NK cells in vitro. Basal cytolytic NK cell activity against tumor cells among peripheral blood mononuclear cells (PBMCs) of routine patients was determined in a standardized NK cell cytotoxicity assay. The impact of cultivation of PBMCs with B. firmus preparation Bacillus firmus e volumine ex muris cellulae (Bacillus firmus (evc)) 6x on tumor cell killing by NK cells was monitored in relation to basal NK cell activity. This study showed that stimulation of PBMCs with Bacillus firmus (evc) 6x in vitro led to a significant increase in NK cell function. Substantial improvement in cytolytic NK cell activity (more than 1.3-fold of basal activity) was much more pronounced for patients with compromised NK cell function. Due to its immunostimulatory mode of action, Bacillus firmus (evc) may be of particular importance in therapy of patients with NK cell deficiency.
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Killer Cells, Natural , K562 Cells , Bacillus firmus/immunologyABSTRACT
Objective To construct the eukaryotic expression vectors of fibroblast growth factor receptor 3(FGFR3) MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN, and to detect their expressions in human chronic myeloid leukemia(CML)K562 cell line.Methods The full-length FGFR3 (fgfr3-WT)and dominant negative FGFR3 (fgfr3-DN)were amplified by polymerase chain reaction (PCR). The two genes were respectively digested with EcoRⅠand BamHⅠ,and then ligated into MSCV/puro to construct the recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN which were tranduced into K562 cells by LipofectaminTM 2000 after PCR,double digestion and DNA sequencing.The expressions of FGFR3 protein in K562 cells were detected by Western blotting and flow cytometry. Results The recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN were amplified by PCR method, and the results showed fgfr3-WT of 2 400 bp and fgfr3-DN of 1 200 bp had been successfully cloned into MSCV-puro vector. The 2 400 bp fragment was oblained after double digestion of recombinant plasmid.The sequencing results showed that the size of fgfr3-WT was 2 400 bp which was the same as the sequence from GeneBank.Fgfr3-DN of 1 200 bp was also in conformity with the expected sequence.Compared with control (K562 MSCV)group,the expression level of FGFR3-WT in MSCV/puro-fgfr3-WT transfection (K562-WT)group was increased to above 10 times.There was high expression of FGFR3-DN in MSCV/puro-FGFR3-DN transfection (K562-DN)group,but there was no expressions in control(K562 MSCV)group and K562-WT group.The flow cytometry results showed that the high expressions of FGFR3-WT were in 57.5% cells in K562-DN group and the high expressions of FGFR3-DN were in 41.5% cells in K562-DN group. Conclusion The K562 cell lines highly expressing FGFR3-WT and FGFR3-DN are constructed successfully.
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Background: To assess the increased cellular immunity of Peripheral Blood Mononuclear Cells (PBMC) derived LAK cells from endometriosis patients towards endometriosis cell cultures after stimulation with IL-2. Methods: This study is a quasi-experimental study of pre and post treatment using controls. Phenotype evaluation of CD3+CD4+, CD3+CD8+ and CD56+ effector cells of PBMC from endometriosis patients and controls was performed. Cytotoxicity test of PBMC from endometriosis patients and control towards Daudi, K562 cell line and endometriosis cell cultures using 51Chromium release assay was also carried out. Results: Phenotype evaluation of PBMC from endometriosis patients (n = 10) and controls (n = 6) were done prior to and after IL-2 stimulation. Before IL-2 stimulation, CD3+CD4+, CD56+ from endometriosis group (n = 10) tend to be lower than control (n=6) whereas CD3+CD8+ were higher in endometriosis group than controls. After IL-2 stimulation, CD3+ CD8+, CD56+ of PBMC from endometriosis group were significantly increased (p < 0.05). Cytotoxicity test revealed a significant increase (p < 0.05) in both PBMC’s effector cells from endometriosis and control group towards target cells, Daudi, and K562 cell lines after IL-2 stimulation. PBMC’s effector cells cytotoxicity from both endometriosis and control towards target endometriosis cell cultures were also elevated after IL-2 stimulation. Conclusion: LAK cells derived IL-2 stimulated PBMC from endometriosis patients increased cellular immunity towards endometriosis cell cultures.
Subject(s)
Endometriosis , Killer Cells, Lymphokine-ActivatedABSTRACT
Objective To explore the effects and the possible molecular mechanism of flavonoids of puerarin (PR) on chronic myelogenous leukemia (CML) cell line K562 and acute promyelocytic leukemia (APL) cell line NB4 in vitro. Methods MTT assays were used to detect the inhibitory effects of cell proliferation. The apoptosis of K562 and NB4 cells was detected by flow cytometry marked with Annexin V/PI. The expression of bcr-abl, p53, bcl-2, Fas/FasL in K562 cells and JNK, PARP, bcl-2 and Caspase 3 in NB4 cells at protein level was detected by Western blot. Results PR could inhibit the proliferation of K562 and NB4 cells in a time-dose dependent manner. The expression of protein levels of bcr-abl fusion gene declined, while the p53 protein otherwise increased, and both were in a dose-dependent manner (F = 18.74, P <0.05). The application of PR had no effect on bcl-2 and Fas/FasL protein expression in K562 cells. The JNK, PARP and Caspase3 proteins were upregulated in NB4 cells, while bcl-2 was downregulated with the increasing concentrations of PR (F=42.32, P <0.05). Conclusion PR could inhibit leukemic cell proliferation, induce cell cycle block, and increase cell apoptosis through different molecular mechanisms. It suggestes that PR might potentially be a kind of broad spectrum anti-leukemia agent.
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Objective To observe anti-sense phosphorothioate oligonucletide (ASPSODN) targeted directly to hTERT mRNA to its inhibiting effect on aimed gene and the influence on the telomerase activity, cellular proliferation, cell apoptosis of K562 cells. Methods Human leukemia cell line K562 was transfected with anti-sense oligonucleotide ASPSODN by liposome. The proliferation activity of K562 cell line was determined by using methyl thiazolyl tetrazolium assay, and telomerase activity was detected by TRAP-PCR-ELISA. Flow cytometry was adopted to examine apoptotic rate and cell cycle. RT-PCR was used to detect the expression of target gene hTERT mRNA. Results 0.6 μmol/L ASPSODN (0.42 ±0.16) was remarkably decreased the expression of hTERT mRNA, Telomerase relative activation was decreased by 52 %. According to 0.6 (μmol/L ASPSODN caused significant the inhibition of K562 cell growth. Apoptotic rate and cell cycle was examined by 0.6 μmol/L ASPSODN with flow cytometry. The cell apoptosis rate of 0.6μmol/L ASPSODN were 10.31 %. It showed the cells treated with 0.6 uU PSASODN arrested in G_1/C_0. The ratio of cells in G_2/M and S period was reduced. But there was no characteristic apoptosis peak. Conclusion ASPSODN targeted hTERT can inhibit the expression of target gene hTERT mRNA, and decrease the telomerase activity of K562 cells. ASPSODN can inhibit strongly the proliferation of K562 cell and induce cell apoptosis by decreasing telomerase activity.
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Purpose To investigate the effects and the mechanism of oleum curcumae wenchowensis (OCW) with different concentrations (0, 2.5, 5, 10 and 20 mg/mL) on chronic myeloid leukemia cell line K-562 cells in vitro.Methods The apoptosis of K-562 cells was dyed by Hoechest 33258 and detected by flow cytometry marked with Annexin V/PI. The Expression of Fas/FasL, bcr/abl, bcl-2 and p53 was detected by semi-quantity reverse transcript-polymerase chain reaction (RT-PCR) and Western blot.Results The results showed that the apoptosis rates were gradually elevated. The expression of Fas and FasL protein was increased in a concentration dependent manner, while bcr/abl, bcl-2 and p53 had no significant changes.Conclusion OCW could induce the apoptosis of K-562 cells by up-regulating the expression of Fas/FasL protein.
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AIM: To explore the different effect and mechanism of arsenic sulfide on telemorase activity and hTERT-mRNA expression in CML cell lines-KS62 and APL cell lines-NB4. METHODS: Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). The expression of hTERT-mRNA was analyzed by semi-quantitative RT-PCR. Flow cytometry was used to analyze the cell cycle and apoptosis. RESULTS: 0.15-0.6 mg/L arsenic sulfide (72 h)can induce apoptosis and inhibit telomerase activity and hTERT-mRNA expression in NB4 cell. The concentration of arsenic sulfide with the same effect on K562 cell was 0.3-3 mg/U 0.3 mg/L arsenic sulfide (72 h) can cause the proportion of the NB4 cell in G2/M phase increased, but for K562 cell, The concentration of arsenic sulfide was 1.5 mg/L. CONCLUSION: Telomerase system may be one.of the pathway for arsenic sulfide inducing apoptosis of NB4 and K562 cell; G2/M phrase arrest may have correlation with decrease of telomerase activity; The sensitivity of NB4 and K562 call for arsenic sulfide is different, the mechanism of it need to study more.
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Objective To study the effects of the RNA secondary structure on the RNA interference efficacy and to optimize the RNAi target sequence selection.Methods N-Ras mRNA in K562 human chronic erythro-leukemia cell line was selected as the target for RNAi experiments.Four siRNAs were designed aiming at secondary structure region and none-secondary structure region separately.The N-Ras expression change and the cell growth was tested by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR),light microscopy,MTT,and flow cytometry.Results Knock-down efficiency was recorded in all of the 4 experiment groups,but the degree varied a lot.The siRNAs targeting none-secondary structure region presented higher silencing efficiency than those targeting secondary structure region.Conclusion The secondary structure of mRNA closely relate to the RNA interference efficiency,which should be considered in the processing of the target sequence selection.
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Aim To explore the effect of 4-amino-2-trifluoromethyl-phenyl retinate(ATPR)on proliferation,differentiation activity in K562 cell line,and to research the mechanisms.Methods Cell proliferation was assessed by MTT assay.Cell differentiation index was analyzed by NBT reduction test.Morphologic changes were observed by Wright's staining in the light microscope. Cell cycle was determined by FCM.The mRNA expression of Cyclin D1,Cyclin E,CDK2,CDK4,CDK6,P21cip1,P27kip1,P57kip2,PCNA mRNA were detected by RT-PCR.While the protein expression of cyclin D1 and CDK4 was detected by Western blot.Results The growth of K562 cells was inhibited in a dose-dependent manner.NBT reduction test indicated that the ATPR could induce differentiation of K562 cells and increase the positive cell ratio.Morphologic changes were observed after Wright's staining using inverted phase contrast microscope.The proportion of cells in G0/G1 phase increased while S phase cells decreased.Cell cycle progression was blocked in the G1 phase.The expression of Cyclin E,cyclin D1,CDK2,CDK4,CDK6 mRNA decreased,while PCNA,P21 cip1,P27 kip1 change was not obvious,but P57 (kip2) mRNA expression was increased.Cyclin D1 and CDK4 protein expressions were reduced as well.Conclusions ATPR inhibits the growth of K562 cells and induces differentiation.P57 kip2 plays a key role in differentiation.Moreover,high level of P57kip2 is regulated via inhibiting its degradation through reducing proteasome-dependent proteolysis,and ATPR plays a role in cell cycle arrest.
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Aim To investigate effects of 1?,25-Dihydroxyvitamin D_3 on the growth inhibition,proliferation of K562 cells and to explore its mechanism. Methods The expression of vitamin D receptor(VDR) was indentified by indirect immunofluorescent stainings.Cell growth,proliferation,apoptosis and cells cycle were evaluated by MTT assay,acridine orange/ethidium bromide(AO/EB) and flow cytometry PI staining.The enzymatic activity of the caspases-3 class of K562 cells was determined by colorimetric assay.Results ① VDR was present in K562 cell nucleus;② 10~(-8) mol?L~(-1) 1,25(OH)_2D_3 could markedly inhibit K562 cells growth and induce cells apoptosiswith most of cells being arrested in G_2/M phase.The ratio of apoptosis increased from 4.1%(control group) to 26.5%(treatment group),P
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BACKGORUND: Trauma, surgical stress, and anesthesia are often associated with postoperative immune suppression and an increased susceptibility to infection. The role of propofol in a patient who may be at the risk of impaired immune function is contradictory. To access the possible role of propofol on human immune function, we investigated the cytotoxic activity of mononuclear cells from peripheral blood. METHODS: Healthy human mononuclear cells (MNCs) were isolated and stimulated with lipopolysaccharide (LPS) for 5 hrs. Activated MNCs were cultured in the presence of varying concentrations of propofol for 20 hrs and lactate dehydrogenase (LDH) release was measured to evaluate NMC cytotoxicity against K-562 cell target cells (cell to target 40:1). RESULTS: Propofol exposure at concentrations of 1, 5 and 10mug/ml did not significantly affect LDH release from K-562 cells, but the cytotoxic activity of MNCs was significantly suppressed at a concentration of 50mug/ml. (P<0.01) CONCLUSiONS: Since the concentrations of 1, 5 and 10mug/ml of propofol are in the clinically acceptable range for sedation and anesthesia, this result suggest that propofol does not significantly alter the cytotoxicity of NMCs in septic conditions.
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Humans , Anesthesia , L-Lactate Dehydrogenase , PropofolABSTRACT
Objective To study the effect of tetrandrine on K 562 cells and its possible mechanism. Methods Light microscope,electron microscope and immuno fluorescence staining were used to detect the morphological changes of K 562 cells,to analyse the cell cycle of K 562 cells FCM was performed,to detect the expression of Bcl 2 gene and wild type p53 gene in K 562 cells ABC method was carried out,and to detect in cell death TUNEL method was applied. Results K 562 cells treated with tetrandrine for 48?h showed early changes of apoptosis.DNA synthesis was reduced.Bcl 2 expression was decreased while wild type p53 gene expression was increased.TUNEL showed DNA breakage.Conclusion\ Tetrandrine inhibits the growth of K 562 cells.The effect is related with the concentration used.Tetrandrine can induce apoptosis of the cell.
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Objective To construct the suppression subtractive library of hemin-inducing K562 cells for identifying the cDNA genes of globin synthesis regulatory factors expressed in K562 cells induced by hemin. Methods K562 cells were cultured under different concentration of hemin, both the percentage of positive benzidine staining cells and hemoglobin content were measured, the most reasonable concentration of hemin was chosen for inducing incubation of K562 cells. The poly (A) positive RNA (mRNA) was isolated from the hemin-induced K562 cells (tester) and non-induced K562 cells (driver) respectively, and double-strand cDNA molecules were synthesized by reverse transcription. After two times subtractive hybridization followed by two times polymerase chain reaction (PCR) amplification, the forward subtracted PCR products were ligated with pGEM T-Easy vector and the subtracted cDNA library was constructed. The library clones were selected by blue-white screening. The plasmid DNAs of the single positive colony were purified and digested by EcoRⅠ, and the inserts in plasmid were amplified by PCR. Results The maximum of positive benzidine staining cells percentage and hemoglobin content of K562 cells were obtained in 50?mol/L of hemin inducing condition. The suppression subtractive library of hemin-inducing K562 cells was constructed after K562 cells were induced by 50?mol/L of hemin. The library identification showed that the positive clones contain inserts in different length respectively. Conclusions The suppression subtracted cDNA library of hemin-inducing K562 cells were successfully constructed. This subtracted cDNA library combined with bioinformatics analysis can be used to explore structures and functions of hemin-inducing expression genes in K562 cells.
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Objective: To study the effect of combining bcr-abl Aspo and c-myb Aspo on K562 cells. Methods: Cellswere exposed to oligomers. Cell inhibitory rate was determined by typan blue dye exclusion. CFU-K562 cells were culturedin 0.8% methyleellulose. P210 was measured by flow cytometry. Cellular bcr-abl mRNA was detected by RT-PCR semiquantitative analysis. Cell apoptosis was measured by flow cytometry and observed by electron microscope. Results: When the concentration of both bcr-abl Aspo and c-myb Aspo was 5 μmol/L, K562 cells were still growth in clone state. The growth inhibitory rate was 61.7% at 120 h. P210 was depressed at 24 h and went up to 25.7% at 120 h. The apoptosis rate was 22.5%. While K562 cells were dealt with 10 μmol/L bcr-abl Aspo and 10 μmol/L c-myb Aspo, the cells were growth in dispersal. The cell growth inhibitory rate reached to 92.2% and 64.3% of K562 cells were induced to apoptosisat 120 h. P210 was complelely depressed untill 120 h. The decrease of bcr-abl mRNA was from 69.2% to 85.3% after incubation 48 h with 5 μmol/L Aspo and 10 mol/L. Conclusion: It emerges coordination to combine bcr-abl Aspo and c-myb Aspo on K562 cells, and enhances the anti-leukemia effect.
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Objective: To study the effect of combining bcr abl Aspo and c myb Aspo on K562 cells. Methods: Cells were exposed to oligomers. Cell inhibitory rate was determined by typan blue dye exclusion. CFU K562 cells were cultured in 0.8% methylcellulose. P210 was measured by flow cytometry. Cellular bcr abl mRNA was detected by RT PCR semiquantitative analysis. Cell apoptosis was measured by flow cytometry and observed by electron microscope. Results: When the concentration of both bcr abl Aspo and c myb Aspo was 5 ?mol/L, K562 cells were still growth in clone state. The growth inhibitory rate was 61 7% at 120 h. P210 was depressed at 24 h and went up to 25.7% at 120 h. The apoptosis rate was 22.5%. While K562 cells were dealt with 10 ?mol/L bcr abl Aspo and 10 ?mol/L c myb Aspo, the cells were growth in dispersal. The cell growth inhibitory rate reached to 92.2% and 64.3% of K562 cells were induced to apoptosis at 120 h. P210 was complelely depressed untill 120 h. The decrease of bcr abl mRNA was from 69.2% to 85.3% after incubation 48 h with 5 ?mol/L Aspo and 10 ?mol/L. Conclusion: It emerges coordination to combine bcr abl Aspo and c myb Aspo on K562 cells, and enhances the anti leukemia effect.
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Objective:To Amplify,purify and identify the cDNA library of human K562 cell in E.coli DH5? and to transform it into yeast cell for subsequent target protein screening.Methods:The cDNA library of human K562 cell was amplified.Then the library plasmids were extracted and transformed into Y187 yeast cell.The results of transformation were verified by PCR and restriction enzyme analysis.Results:The cDNA library of human K562 cell was amplified successfully and the diversities were verified.The cDNA library of human K562 cell was also successfully transformed into Y187 yeast cell.Conclusion:Successfully amplification,purification and identification of the cDNA library of human K562 cell lay the foundation for the subsequent protein screening.