Effect of polydatin on the proliferation and apoptosis of THP-1 cells and the mechanism / 中国当代儿科杂志

OBJECTIVES@#To explore the effect of polydatin on the proliferation and apoptosis of acute monocytic leukemia cell line THP-1 and the possible mechanism.@*METHODS@#After THP-1 cells were treated with polydatin at gradient concentrations for 24 hours and 48 hours, their proliferation was determined by CCK-8 assay, and half maximal inhibitory concentration (IC50) was calculated. Logarithmically growing THP-1 cells were divided into two groups, a polydatin treatment group (treated with IC50 of polydatin) and a blank control group (treated without polydatin solution), and incubated for 48 hours. Cell apoptosis and cell cycle were measured by flow cytometry. The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins were measured by Western blotting.@*RESULTS@#After treatment with polydatin, the proliferation of THP-1 cells was strongly inhibited, and the IC50 at 48 hours was 1 800 μmol/L. After treatment with 1 800 μmol/L polydatin solution for 48 hours, the apoptosis rate of THP-1 cells increased significantly compared with the blank control group (P<0.05). The cell cycle was arrested in the G0/G1 and S phases, with a significantly increased proportion of cells in the G0/G1 phase and a significantly decreased proportion of cells in the S phase, as compared with the blank control group (P<0.05). The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins decreased significantly compared with the blank control group (P<0.05).@*CONCLUSIONS@#Polydatin can effectively inhibit the proliferation, block the cell cycle, and induce the apoptosis of THP-1 cells, which may be related to inhibition of the PI3K/AKT/mTOR signaling pathway.

Polarization of human acute monocytic leukemia THP-1 cells-derived macprophages induced by Nippostrongylus brasiliensis proteins in vitro / 中国血吸虫病防治杂志

Objective To investigate the polarization of human acute monocytic leukemia THP-1 cells-derived macrophages induced by Nippostrongylus brasiliensis proteins in vitro, so as to provide insights into the elucidation of the mechanisms underlying host immune responses to hookworm infections. Methods The in-vitro culture of N. brasiliensis was established and maintained in the laboratory, and the third- (L3) and fifth-stage larvae (L5) were collected under a sterile condition for preparation of L3 and L5 proteins. The in-vitro culture of THP-1 cells was established, stimulated with 500 ng/mL PMA to yield M0 macrophages that were adherent to the plate wall. The LPS + IFN-γ group, IL-4 + IL-13 group, L3 protein group and L5 protein group were given stimulation with 500 ng/mL LPS plus 100 ng/mL IFN-γ, IL-4 and IL-13 (both 100 ng/mL), L3 protein (5 mg/mL) and L5 protein (5 mg/mL), respectively, while the negative control group was given no stimulation. The cell morphology was observed using microscopy, the mRNA expression of M1/M2 macrophages-specific genes was quantified using a quantitative real-time PCR (qPCR) assay, and the surface markers of M1/M2 macrophages were detected using flow cytometry, while the levels of cytokines secreted by M1/M2 macrophages were measured using enzyme-linked immunosorbent assay (ELISA) following stimulations, so as to examine the polarization of THP-1-derived macrophages induced by N. brasiliensis proteins in vitro. Results Following stimulation with PMA, THP-1 cells appeared wall-adherent M0 macrophages, and polarized to typical M1 macrophages following stimulation with LPS + IFN-γ, and typical M2 macrophages following stimulation with IL-4 + IL-13, IL-3 protein or L5 protein. There was a significant difference in the proportion of M1 macrophages among the negative control group, the LPS + IFN-γ group, the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (χ2 = 3 721.00, P < 0.001), with the highest proportion detected in the LPS + IFN-γ group, and there was also a significant difference in the proportion of M2 macrophages among groups (χ2 = 105.43, P < 0.001). There were significant differences among groups in terms of the mRNA expression of CCL2 (F = 191.95, P < 0.001), TNF-α (F = 129.95, P < 0.001), IL-12b (F = 82.89, P < 0.001), PPARγ (F = 11.30, P < 0.001), IL-10 (F = 9.51, P < 0.001) and Mrc1 genes (F = 12.35, P < 0.001). In addition, there were significant differences in the proportion of positive CD86 and CD206 expression among groups (χ2 = 24 004.33 and 832.50, P < 0.001). Higher IL-1β and TNF-α levels were measured in the LPS + IFN-γ group than in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (P < 0.001), and greater TGF-β1 and IL-10 levels were seen in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group than in the negative control group and the LPS + IFN-γ group (P < 0.05). Conclusions Both L3 and L5 proteins of N. brasiliensis may induce the polarization of THP-1-derived macrophages to M2 type in vitro.

Effect of Arsenic Trioxide Combined with Dihydroartemisinin on THP-1 Cells Proliferation and Apoptosis / 中国实验方剂学杂志

Objective:To investigate the effects of arsenic trioxide combined with dihydroartemisinin on proliferation, cell cycle, and apoptosis of THP-1 cells, and explore the mechanism. Method:The thiazolyl blue （MTT） method was applied to detect the effect of different concentrations of arsenic trioxide, dihydroartemisinin and arsenic trioxide combined with dihydroartemisinin on the proliferation of THP-1 cells. Annexin V/propidium iodide（PI）assay was used to detect the change of THP-1 cell cycle and apoptosis．Western blot was performed to assess the expression of cysteine protease-3（Caspase-3）, cleaved Caspase-3, B-lymphocytoma-2（Bcl-2） and Bcl-2 associated X protein （Bax）. The changes of cell morphology were observed under high intension microscope. Result:Compared with blank group, arsenic trioxide and dihydroartemisinin both exhibited obvious antiproliferative effect on the human acute monocytic leukemia cell line THP-1 in time-dose dependence （P<0.01）. After 48 h, compared with the same dose of arsenic trioxide or that of dihydroartemisinin alone, the inhibition effect of 1 µmol·L-1 arsenic trioxide combined with 2 µmol·L-1 dihydroartemisinin on proliferation of THP-1 cells was significantly stronger （P<0.01）. Compared with the control group, arsenic trioxide combined with dihydroartemisinin significantly arrested the cells in G1 phase （P<0.01）, induced the downregulation of Caspase-3 and Bcl-2 （P<0.01） and upregulation of cleaved Caspase-3 significantly（P<0.05）. Conclusion:Arsenic trioxide combined with dihydroartemisinin can significantly inhibit the proliferation and induce apoptosis of THP-1 cells. The possible mechanism may be related to arrest the cells in G1 phase, reduce the expression of Caspase-3 and Bcl-2, increase the expression of cleaved Caspase-3.

Acetoacetate extract of pleione bulbocodioides (franch. ) rolfe induces apoptosis of human leukemia thp-1cells through a mitochondria-regulated intrinsic apoptotic pathway / Extrato de acetoacetato de Pleione bulbocodioides (Franch. ) Rolfe induz a apoptose de células THP-1 de leucemia humana através de uma via apoptótica intrínseca regulada por mitocôndrias

The tubers of three orchidaceous plants, includingPleione bulbocodioides (Franch.) Rolfe, have been used as 'Shan-Ci-Gu' in traditional Chinese medicine for the treatment of bacterial infections and cancers for thousands of years. In this study, the effects of an acetoacetate (EtOAc) extract of P. bulbocodioides on the cell viability and apoptosis of THP-1 (human acute monocytic leukemia cell line) cells and its interaction with possible apoptotic pathways were investigated. THP-1 cells were treated with the EtOAc extract of P. bulbocodioides at different concentrations. The results showed that THP-1 cell viability was significantly inhibited by the EtOAc extract ofP. bulbocodioides with an IC50 of 51.37±2.68 µ g/ mL at 24 h. The examination of cytotoxic effects on healthy cells showed that the EtOAc extract of P. bulbocodioidesdid not show any effect on healthy Vero cells. Selectivity indexes were greater than 15.57, suggesting that the EtOAc extract of P. bulbocodioides had selective toxicity against THP-1 cells. The results of annexin V-FITC/PI and DAPI staining showed that the EtOAc extract of P. bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased in the treatment groups compared with that in the control group (P<0.05). The distribution of cells in the G2 phase of the cell cycle increased along with typical cell apoptosis-induced morphological changes. The levels of the pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 increased with increasing concentration of acetoacetate extract of P. bulbocodioides, while the anti-apoptosis protein Bcl-2 was downregulated. Cyt c and AIF, which are characteristic proteins of the mitochondria-regulated intrinsic apoptosis pathway, also increased in the cytosol with increasing concentrations of the EtOAc extract of P. bulbocodioides. These results showed that the EtOAc extract of P. bulbocodioidessignificantly inhibits cell viability and induces cell apoptosis in the human leukemia cell line THP-1 through a mitochondria-regulated intrinsic apoptotic pathway

Os tubérculos de três plantas orquidáceas, incluindo Pleione bulbocodioides (Franch.) Rolfe, têm sido usados como "Shan-Ci-Gu" na medicina tradicional chinesa para o tratamento de infecções bacterianas e cânceres por milhares de anos. Neste estudo, os efeitos de um extrato de acetoacetato (EtOAc) de P. bulbocodioides na viabilidade celular e apoptose de células THP-1 (linhagem celular de leucemia monocítica aguda humana) e sua interação com possíveis vias apoptóticas foram investigados. As células THP-1 foram tratadas com o extrato EtOAc de P. bulbocodioides em diferentes concentrações. Os resultados mostraram que a viabilidade das células THP-1 foi significativamente inibida pelo extrato EtOAc de P. bulbocodioides com IC50 de 51,37 ± 2,68 µ g/mL às 24 h. O exame dos efeitos citotóxicos em células saudáveis mostrou que oextrato de EtOAc de P. bulbocodioides não mostrou nenhum efeito sobre células Vero saudáveis. Os índices de seletividade foram maiores que 15,57, sugerindo que o extrato de EtOAc de P. bulbocodioides teve toxicidade seletiva contra as células THP-1. Os resultados da coloração da anexina V-FITC/PI e DAPI mostraram que o extrato de EtOAc de P. bulbocodioides induziu a apoptose celular de maneira dose-dependente. A taxa de apoptose foi aumentada nos grupos de tratamento em comparação com o grupo controle (P <0,05). A distribuição de células na fase G2 do ciclo celular aumentou juntamente com alterações morfológicas típicas induzidas pela apoptose celular. Os níveis das proteínas pró-apoptóticas Bax, PARP clivada e caspase-3 clivada aumentaram com o aumento da concentração do extrato acetoacetato de P. bulbocodioides, enquanto a proteína anti-apoptose Bcl-2 foi regulada negativamente. Cyt c e AIF, que são proteínas características da via de apoptose intrínseca regulada por mitocôndrias, também aumentaram no citosol com concentrações crescentes do extrato de EtOAc de P. bulbocodioides. Estes resultados mostraram que o extrato de EtOAc de P. bulbocodioides inibe significativamente a viabilidade celular e induz a apoptose na linha celular de leucemia humana THP-1 através de uma via apoptótica intrínseca regulada por mitocôndrias.

Screening of the anti-atherogenic drugs by using THP-1-HIF-1α-HER-Luciferase cell model / 上海交通大学学报（医学版）

Objective: To screen the anti-atherosclerosis (AS) activity of the compounds by using THP-1-HIF-1α-HER-Luciferase high-throughput model, and to verify the anti-AS function of the effective compounds. Methods: THP-1-HIF-1α-HER-Luciferase cells were pretreated with different concentrations of compounds (1, 10, and 100 μg/mL) for 2 h, then cultured under hypoxia for 24 h. Luciferase activity of cells was detected and compounds with anti-AS activity were screened by luciferase activity evaluation. THP-1 and U937 cells were pretreated with effective compounds, and then induced for 24 h by oxidized low density lipoprotein (OX-LDL). The formation of foam cells was observed by oil red staining. The mRNA level of hypoxia-inducible factor 1α (HIF-1α) was detected by real-time quantitative PCR (qPCR). HIF-1α protein expression was detected by Western blotting. Anti-AS activity of effective compounds were evaluated. Results: Among the 200 compounds, 11 compounds could significantly inhibit the increase of luciferase activity in THP-1-HIF-1α-HER-Luciferase cells induced by hypoxia (all P<0.05), and compound numbered 14 (C14) had the most significant inhibitory effect. THP-1 and U937 cells formed foam cells induced for 24 h by OX-LDL. However, cells were pretreated with C14 for 2 h, which could significantly inhibit the formation of foam cells induced by OX-LDL. Cells were induced for 24 h by OX-LDL, which could significantly increase the expression of HIF-1α mRNA and protein (all P<0.05), while cells pretreated with C14 could significantly inhibit the increase of HIF-1α mRNA and protein expression in a gradient-dependent manner (all P<0.05). Conclusion: THP-1-HIF-1α-HER-Luciferase high-throughput model can be reliability used in screening of compounds with anti-AS activity. C14 has the good anti-AS activity characteristics.

Preparation and identification human anti-Siglec-9 Fab fragment antibody / 中国免疫学杂志

Objective:The prokaryotic expression vector of human anti-Siglec-9 Fab fragment antibody was constructed and purified,while was identified. Methods:The variable and conserved regions of heavy chain and light chain were obtained by polymerase chain reaction respectively(PCR),which was combined by overlap extension PCR and was digested with restriction enzyme,and then it was transformed into Escherichia coli BL21 and was purified by His-trap Lambda Fab column and AKTA system. SDS-PAGE,ELISA and Western blot were used for the identification of human anti-Siglec-9 Fab fragment antibody. The effect of human anti-Siglec-9 Fab fragment antibody on regulating the mRNA expression of TNF-α,IL-1,IL-6,IL-8 was detected by real-time PCR. Results:Successfully obtained the chains of heavy and light, while constructed an activation human anti-Siglec-9 Fab fragment antibody which could specifically bind to Siglec-9 protein. The human anti-Siglec-9 Fab fragment antibody could specifically bind to Siglec-9 was confirmed by SDS-PAGE,ELISA and Western blot. The human anti-Siglec-9 Fab fragment antibody inhibited the mRNA expression of TNF-α,IL-1, IL-6,IL-8. Conclusion:Successful prokaryotic expression,purification,character analysis,and suppressed the mRNA expression of in-flammatory cytokines with the human anti-Siglec-9 Fab fragment antibody and lay the biology foundation for the further study.

Predicting the sensitizing potency of trichloroethylene by in vitro test / 中国职业医学

OBJECTIVE: To predict the sensitizing potency and optimal sensitization dose of trichloroethylene( TCE) by an in vitro skin sensitization test on a human acute monocytosis cell line( THP-1).METHODS: THP-1 cells were cultured in vitro and exposed to 2,4-dinitrochlorobenzene( DNCB),sodium dodecyl sulfate( SDS),tert-butylhydroquinone( tBHQ)and TCE for 24 hours.Flow cytometry was used to detect the expression of cell surface marker such as cluster of differentiation( CD) 86 and CD54,and the optimal dose range for sensitization detection was determined.With the relative fluorescence intensity( RFI),CD86 ≥ 150 and CD54 ≥ 200 as the standard,the sensitizing potency and optimal sensitization dose of TCE were predicted.RESULTS: The concentration range of reagents for sensitization test on THP-1 cells was the dose range at which the relative cell survival rate reached 75.0%-100.0%.DNCB at the doses of 20.83,25.00 and 30.00 μmol/L,tBHQ at the dose of 5.80 μmol/L,TCE at the doses of 8.33,10.00 and 12.00 mmol/L,can cause sensitivity.SDS was recognized as a negative sensitizer.The expression of CD86 and CD54 was the highest when the concentration of TCE was 8.33 mmol/L,which was considered as the best sensitization dose.CONCLUSION: The optimum sensitization dose of TCE is 8.33 mmol/L,which can provide the basis for dose design in future study of TCE sensitization pathways.

Analysis of IL-8 secretion in THP-1 cells stimulated by lipopolysaccharide and capsular polyssacharide extracted from Porphyromonas gingivalis / 上海交通大学学报（医学版）

Objective·To isolate lipopolysaccharide (LPS) and capsular polysaccharide (CPS) from Porphyromonas gingivalis (Pg) and analyze their ability to induce THP-1 cells to secrete interleukin 8 (IL-8) in vitro. Methods·LPS and CPS were isolated from the polysaccharides by gel filtration chromatography, which had already been extracted from Pg ATCC33277 and SJD4. The isolated LPS and CPS from different strains were used to stimulate THP-1 cells in different doses and for different periods. The level of IL-8 in the cell culture suspension was measured by ELISA. Results·LPS and CPS were successfully separated from the polysaccharides in Pg ATCC33277 and SJD4. The relative molecular masses of LPS and CPS were 20000-50000 and 150000-500000, respectively. LPS and CPS could induce THP-1 cells to produce IL-8 in vitro in a time- and dose-dependent manner. The effect of LPS from ATCC33277 on secretion of IL-8 was greater than that of CPS with the dose of 0.1 μg/mL (P<0.05). However, the difference could only be found at 24 h in SJD4, and the effect of CPS was greater (P<0.05). Conclusion·As a component of polysaccharides extracted from Pg, CPS can induce THP-1 cells to secrete IL-8 in vitro in a time- and dose-dependent manner.

An in vitro skin sensitization test based on THP-1 cell line / 中国比较医学杂志

Objective To establish an in vitro skin sensitization test,human cell line activation test (h-CLAT),based on THP-1 cell line (a human acute monocytic leukemia cell line),and to assess the sensitizing potency of plant raw materials of chemical and cosmetic products by this in vitro skin sensitization test.Method THP-1 cells were cultured in vitro and exposed to 11 reference skin sensitization chemicals and 9 samples,by monitoring the cell viability,cell surface marker CD54 /CD86 and relative fluorescence intensity of cells surface after the cells was exposures to the substances,and to discover whether there is a positive reaction.At the same time,Buehler test was used to validate the results of samples tested by h-CLAT.Results 11 reference chemicals were distinguished correctly by h-CLAT.Among the 9 samples tested,7 samples were recognized as negative sensitizer and 2 plant extracted substances were identified as suspicious skin sensitizer.The qualitative classification of the 9 samples by h-CLAT test was consistent with the results obtained by animal test.Conclusions The h-CLAT-in vitro test can be used to replace some animal tests for the prediction of soluble skin sensitizing substances.

Cloning, expression and biological activity of Mycobacterium tuberculosis fusion antigen ESAT-6 and CFP-10 / 军事医学

Objective To clone and express Mycobacterium tuberculosis fusion antigen ESAT-6 and CFP-10 ( EC) and to evaluate the biological activity of the fusion antigen EC in inducing specific cytokines secretion from THP -1 cells. Methods The fusion antigen EC gene was cloned into pET-30 a prokaryotic expression vector and expressed highly in E.coli BL21.Then, the THP-1 cells were stimulated with purified fusion antigen EC of different concentrations (10 and 20 μg/ml).Culture supernatants were collected after 12 h and 24 h, respectively.The secretion levels of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-αand IFN-γin THP-1 cell culture supernatants were detected using Bio-PlexProTM Assays kit.Results The M.tuberculosis fusion antigen EC was cloned and expressed successfully .The secretion levels of IL-6, IL-8 and TNF-αin EC infected THP-1 cells were significantly higher than those in THP-1 cells (P<0.05).The secretion levels of other cytokines did not change significantly .Conclusion The obtained M.tuberculosis fusion antigen EC has biological activity in inducing the THP-1 cells to secrete a higher level of IL-6,IL-8 and TNF-α.

Construction and identification of Mcl-1 gene shRNA expression plasmid in targeted silenced macrophages / 西安交通大学学报(医学版)

Objective To study the expressions of myeloid cell leukemia-1 (Mcl-1 ) gene in mouse macrophages Raw264.7 and human macrophages THP-1,to screen out the cell lines with high levels of expression as the experimental cells,and based on the screening results to construct the short hairpin RNA(shRNA)eukaryotic expression plasmid targeting mice Mcl-1 gene for transfection and further screen out the shRNA expression plasmid with the most obvious effect in silencing Mcl-1 gene.Methods Semi-quantitative PCR method was used to detect the expression of Mcl-1 mRNA in the two kinds of macrophages.Western blotting was adopted to detect the expressions of Mcl-1 proteins in the two kinds of macrophages.Three different gene loci for Mcl-1 shRNA fragments were designed with small molecules interfering RNA (siRNA)software.Eukaryotic expression plasmid Mcl-1 shRNA 1-3 carrying the shRNA fragments was constructed by a company.And the eukaryotic expression plasmid vector was transfected into scavenger cells Raw264.7 mice via through the liposome.The transfection results 24 h and 48 h after transfection were observed under the inverted fluorescence microscope;Mcl-1 mRNA and protein expression were detected by real time quantitative PCR and Western blot,respectively.Results The relative expression levels of Mcl-1 mRNA and protein in mouse macrophages Raw264.7 were significantly higher than those of human macrophages (P <0.05).The shRNA expression vector constructed within 24 h and 48 h could decrease Mcl-1 mRNA and protein levels in Raw264.7 cells,especially with the most obvious silencing effect at 48 h.The 48-h transfection group differed significantly from normal group,liposome group and negative control group (P <0.05).Compared with Mcl-1shRNA1and Mcl-1shRNA2,Mcl-1shRNA3 had the strongest effect in silencing Mcl-1mRNA and protein.Conclusion We have successfully screened out experimental Raw264.7 cells and Mcl-1 shRNA eukaryotic expression plasmid which has an obvious silencing effect targeting on Mcl-1 in mice macrophages Raw264.7.

Effecst of let-7e on the cell activities of monocytic cell line THP-1 and the possible mechanism / 中华微生物学和免疫学杂志

Objective To investigate the effects of a microRNA family member , let-7e, on mono-cytic cell line THP-1 with regard to cell apoptosis and cytokine secretion and to analyze the possible mecha -nism.Methods THP-1 cells were transfected with mimic negative control (cy3) and observed with immu-nofluorescence microscopy for the evaluation of transfection rate .The expression of let-7e in THP-1 cells re-spectively transfected with let-7e mimic, mimic negative control, let-7e inhibitor and inhibitor negative con-trol were detected by qRT-PCR.MTT assay and flow cytometry analysis were used to detect the activities and apoptosis of transfected THP-1 cells.Western blot assay was performed to measure the expression of the genes encoding interferon alpha-inducible protein 6( IFI6 ) , enhancer of zeste homolog 2 (EZH 2 ) and caspase -3 that were target genes of let-7e predicted by bioinformatics analysis .THP-1 cells were transfected with let-7e mimic and mimic negative control for 48 h and then stimulated with LPS for 2 h for further detec-tion.The supernatants of cell culture were collected for the detection of secreted cytokines by Human Cyto -kine Array.Results The monocytic THP-1 cells were transfected with mimic negative control with a trans-fection efficiency of about 75%.There were 8.551±0.365, 83.893±15.941, 38.858±2.743 and 0.594± 0.174, 2.427±1.229, 3.053±0.207 fold increases in let-7e expression after the transient transfection of THP-1 cells with let-7e mimic and let-7e inhibitor for 12 h, 24 h and 48 h, respectively.The transfection of let-7e mimic into THP-1 cells enhanced the cell activities and inhibited the apoptosis of the transfected cells . Bioinformatics analysis showed that let-7e bound to the genes encoding EZH 2, IFI6 and caspase-3 with the mirSVR scores of -0.1608,-0.5693 and-0.9423, suggesting them as the predicted target genes of let-7e. The expressions of IFI6, EZH2 and caspase-3 in let-7e mimic transfected THP-1 cells were decreased as in-dicated by Western blot assay .The results of Human Cytokine Array showed that the expression of LPS-in-duced cytokines including CD154, G-CSF, CD54, IL-13, IL-1RA and IL-23 were inhibited in let-7e mimic transfected THP-1 cells. Con clusion Let-7e had an anti-apoptosis effect on monocytic THP-1 cells and in-fluenced the secretion of LPS-induced cytokines in THP-1 cells.Let-7e might regulate the biological function of THP-1 cells through inhibiting the expression of target genes encoding caspase -3, IFI6 and EZH2.

Sorting,identification and enrichment of side population cells in acute monocytic leukemia THP-1 Cells / 中华实用儿科临床杂志

Objective To assay and determine whether the human acute monocytic leukemia cell line THP-1 contains side populations (SP) cells,and to increase the proportion of SP cells using cytarabine (Ara-C).Methods Fluorescent microscope and flow cytometry (FCM) were employed for detecting the percentage of SP cells in THP-1 cells.Then,SP and non-SP (NSP) subpopulations were collected and identified.Finally,THP-1 cells were incubated with different concentrations of Ara-C for 24 hours and detected the proportion of SP cells,respectively.Results The results demonstrated that the percentage of SP cells was (1.81 ± 0.99) ％ in THP-1 cells.A majority of the SP cells remained in the G0/G1 phase,and the expressions of CD34 + and CD34 + CD38-and the proliferative ability of the SP cells were higher than those of NSP cells (P ＜ 0.05).The mRNA expression of multidrug resistance genes (ABCG2,ABCB1),apoptosis regulation genes (Bcl-2) and the Bcl-2/Bax ratio of SP cells were higher than those of NSP cells.SP cells have been shown to be more tumorigenic than NSP cells.After co-culture with Ara-C,the proportion of SP cells increased significantly and presented in a concentration-dependent manor.Conclusions All of these findings suggest that the THP-1 cell line contains SP cells and the SP cells possess some intrinsic stem cell properties.The proportion of SP cells can be increased when co-cultured with Ara C,and this technique is a useful and important application for the study of LSCs.

Influence of Mycobacterium tuberculosis 19-kDa lipoprotein on function and phenotype of macrophage / 中国人兽共患病学报

To observe the influence of Mycobacterium tuberculosis 19-kDa lipoprotein (Mtb P19) on function and phenotype of macrophage,the Mtb P19 was prepared from cultured Mtb H37Ra and the phorbol myristate acetate-differentiated THP-1 cells were incubated with P19 at the concentration of 10 μg/mL with 5% CO_2 at 37℃ for up to 48 hours.Supernatants were collected for TNF-α and IL-6 detection by ELISA,then the phenotype fluorescent antibodies were stained to analyze HLA-DR expression changes between control group and experimental group.Flow cytometry and microscopy was used to assay the phagocytosis in macrophages stimulated by Mtb P19.IL-6 and TNF-α in collected supernatants were detected.Results indicated that both were found significant increases and their phagocytosis were enhanced.Comparing to the control group,the mean fluorescence intensity showed a significant increase 24hs after stimulation.It presents that Mtb P19 could be able to induce macrophages activation,and it would be significantly important for protection during infection period.

Influence of Mycobacterium tuberculosis 19 kDa lipoprotein upon the expression and distribution of Toll-like receptor-2 on the surface of macrophages / 中国人兽共患病学报

To observe the effect of Mycobacterium tuberculosis 19 kDa lipoprotein (Mtb P19) upon the expression and distribution of Toll-like receptor-2 (TLR-2) on the surface of macrophages, Mtb P19 was prepared from the cultured M.tuberculosis H37 Ra strain , and phorbol myristatye acetate (PMA)-differentiated THP-1 cells were co-cultivated with Mtb P19 at concentration of 10 g/mL and at 37 ℃ temperature and a condition containing 5% CO_2.for 6 hours.. The distribution of TLP-2 on the surface of macrophages was investigated by immuno-cellular chemical method (ICC) while the effect of Mtb P19 upon the expression of TLR on the surface of macrophages was assayed by fluorescent antibody staining. In addition,the changes of TLR-2 expression before and after the effect of Mtb P19 were investigated by flow cytometry analysis and change of TLR-2 arrangement after stimulation with Mth P19 was determined by co-focal microscopy. It was found that the TLR-2 molecules were evenly distributed on the surface of macrophages as demonstrated by ICC. The mean fluorescent intensity increased significantly after stimulation with Mtb P19 for 6 hours in comparison with that of the control group, and the patchy surface with fluorescent staining positive zones could be detected on the surface of macrophages. Nevertheless , the distribution of TLR-2 molecule in the control group appeared to be randomly dynamic. It is evident that the Mth P19 not only can induce the surface expression of TLR-2 molecules, but also cause a functional aggregation of this receptor.

NF-κBp50 is Associated With DC-SIGN Expression Induced by IL-4 in THP-1 Cells / 生物化学与生物物理进展

DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is specific receptor on Dendritic cells, and plays a pivotal role on antigens presentation. Uptodate, the clear regulation mechanisms for DC-SIGN expression are not available.IL-4 is one of the most important cytokines inducing DC-SIGN production, while, NF-κB is an important transcription factor controlling signaling transduction. Both IL-4 and NF-κB are closely related to DC-SIGN regulation. NF-κB and IL-4 actions on DC-SIGN promoter activity, DC-SIGN expression as well as interactions between IL-4 and NF-κB were investigated in THP-1 cell. It was found that the mutation of NF-κB binding site in DC-SIGN promoter results in DC-SIGN promoter activity decrease about 50%.NF-κBp50 stimulates DC-SIGN expression in THP-1 cells. IL-4 upregulates DC-SIGN expression on THP-1 cells as well as NF-κB production. These data reveal that NF-κB is associated with IL-4 induced DC-SIGN expression.

Apoptosis inducing effects of oridonin on THP-1 cells and its mechanisms of action / 肿瘤研究与临床

Objective To investigate the apoptosis inducing effects of oridonin on leukemic THP-1 cells and its mechanisms of action. Methods THP-1 cells in culture medium in vitro were given different concentrations of oridonin (16～56 μmol/L) for 24, 48 and 72 h. The inhibitory rate of the cells were measured by MTT assay, apoptotic morphology was observed by Hoechst 33258 staining, and Annexin V/PI staining was used to detect cell apoptosis by flow cytometry (FCM). Caspase-3 and poly (ADP-ribose) polymerase (PARP) expression were detected by Western blotting. Results Oridonin (over 32 μmol/L) could inhibit the growth of THP-1 cells and cause apoptosis remarkably, the suppression was both in time-and dose-dependentmanner. Marked morphological changes of cell apoptosis such as condensation of chromatin was clearly observed by Hoechst 33258 staining, and Annexin V/PI staining showed that apoptotic cells gradually increased after the cells treated with oridinon. Western blotting showed cleavage of the caspase-3 zymogen protein (32×103), with the appearance of its 20×103 subunit, and a cleaved 89×103 fragment of 116×103 PARP was also found. Conclusion Oridonin can inhibit cell growth and induce apoptosis in THP-1 cells via activation of caspase-3. The results indicated that oridonin might be an important potential anti-leukemia reagent.

CRP promotes lectin-like oxidized low density lipoprotein receptor-1 expression on THP-1 derived macrophages / 基础医学与临床

Objective To explain the effects of C-reactive protein(CRP) on lectin-like oxidized low density lipoprotein receptor-1 expression on THP-1 derived macrophages and the related signal transduction pathways.MethodsTHP-1 cells were differentiated into macrophages with the stimulation of PMA.THP-1 derived macrophages were incubated with CRP and co-incubated with inhibitors of NF-?B、AP-1 and MARK signal transduction pathways.The expression of LOX-1 antigen and mRNA was analyzed by ELISA and RT-PCR.Results CRP stimulated the expression of LOX-1 antigen and mRNA on macrophages in a dose-dependent manner.NF-?B inhibitor BAY11-7085 suppressed the inducible effects of CRP on LOX-1 expression.Conclusion CRP increased LOX-1 expression on THP-1 derived macrophages at transcription and post-transcription levels.The NF-?B signal transduction pathway may be involved in such process.

Correlation between Endotoxin Tolerance in Human Monocyte Leukemia Cell Line THP-1 with Glucocorticoid Receptor-α / 华中科技大学学报（医学）（英德文版）

Human monocyte leukemia cell line THP-1 was stimulated with lipopolysaccharide (LPS) to simulate the sepsis model and the expression of human glucocorticoid receptor-α (GR-α) mRNA in montocytes with endotoxin tolerance was investigated. THP-1 cells were cultured in serum-free medium, randomly divided into groups A, B, C, D and E, and stimulated with 0, 10, 10,100, 0 ng/mL LPS for 24 h followed with 100, 100, 10, 100, 0 ng/mL LPS for another 24 h respectively. The expression of GR-α mRNA was detected by semi-quantitative reverse transcriptional polymerase chain reaction. Tumor necrosis factor-α (TNF-α) was determined by enzyme linked immunosorbent assay (ELISA). The results showed that the A values of GR-α/β-actin in groups A,B, C, D and E was 0. 607±0. 006, 0. 368±0. 005, 0. 484±0. 008, 0. 509±0. 004 and 0. 564± 0. 014 respectively with the difference being significant among the groups (P＜0. 05). The GR-α mRNA expression was negatively correlated with the TNF-α expression (P＜0.01). It was concluded that the down-regulation of the expression of GR-α mRNA in endotoxin tolerance THP-1 cells might play an important role in the development of endotoxin tolerance in THP-1 cells.

Effect of Cadmium on Cytokine Gene Expression in a Human Monocytic Cell Line, THP-1 / 대한산업의학회지

Cadmium, a potent toxic metal, poses a serious environmental threat but the mechanism of its toxicity remains unclear. Also, cadmium is a known immunotoxic agent in animal studies and induces pathophysiological effects by modulating components of immune system. Cytokines are being increasingly recognized as essential mediators of normal and pathologic immune response. Cells of mononuclear phagocytic system are strategically located at portals of entry in humans and therefore may be particularly at risk for cadmium exposure through contaminated air, food, and drinking water. In the present study, we investigated the effect of cadmium cytotoxicity for the monocyte and expression of cytokine gene in the control and cadmium treated human monocytic cell lines using RT-PCR method. The results showed that cadmium inhibited cell proliferation at 0.1mM cadmium treated cells for 24 hours. The TNF-alpha mRNA was expressed in both control and cadmium treated cells but not IL-6 and IL-1 beta The mRNA levels of TNF-alpha were examined during 24 hours culture period, at different time points. The expression of TNF-alpha mRNA increased in both 0.01mM and 0.1mM cadmium treated cells, but did not show dose-response relationship. According to cadmium treated duration, expression of TNF-alpha mRNA was more decreases in 24 hours than 6 hours. The decreased levels of mRNA of TNF-alpha that cadmium suppresses iris production at the transcription level.