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Acute mononuclear leukemia is a type of disease with a high incidence in acute myeloid leukemia. With the continuous deepening of traditional Chinese medicine research, some progress has been made in the research on its clinical efficacy and mechanism of action. This article reviews the research progress in the treatment of acute mononuclear leukemia with traditional Chinese medicine in recent years.
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Objective To investigate the roles of Dectin-1 in phagocytosis of Candida albicans (C.albicans) by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1.Methods THP-1 macrophage-like cells served as the target cells,and were transfected with small interfering RNA (siRNA) targeting Dectin-1 to down-regulate the expression of Dectin-1 receptor (siRNA-Dectin-1 group).THP-1 macrophage-like cells transfected with nonsense siRNA (siRNA-NC) served as a negative control group.After transfection,the THP-1 macrophage-like cells in the above 2 groups were cocultured with heat-killed C.albicans separately.And then,fluorescence microscopy was performed to count THP-1 macrophage-like cells phagocytosing C.albicans,and flow cytometry was used to determine the mean fluorescence intensity (MFI) of dihydrorhodamine (DHR)-123 fluorescent cells.Statistical analysis was done by one-way analysis of variance (ANOVA) and t test with the SPSS19.0 software.Results After transfection with siRNA-Dectin-1,the mRNA and protein expression of Dectin-1 significantly decreased in THP-1 macrophage-like cells (t =26.163,P ＜ 0.001).After 1-,2-,4-hour co-culture of THP-1 macrophagelike cells with C.albicans,fluorescence microscopy showed that the phagocytosis rates of C.albicans by THP -1 macrophage-like cells were significantly lower in the siRNA-Dectin-1 group than in the negative control group (17.5％ vs.22.1％,18.6％ vs.24.3％,39.2％ vs.59.1％,respectively,all P ＜ 0.05),so were the percentage of THP-1 macrophage-like cells phagocytosing more than 3 C.albicans cells (2.2％ vs.4.7％,2.5％ vs.5.4％,5.1％ vs.8.3％,respectively,all P ＜ 0.05).After 30-minute,1-,2-and 4-hour co-culture of THP-1 macrophage-like cells with DHR-123-labelled C.albicans,flow cytometry showed that the MFI of C.albicans-phagocytosing cells was significantly lower in the siRNA-Dectin-1 group than in the negative control group (36.8 vs.45.7,54.3 vs.62.4,72.1 vs.84.9,93.6 vs.116.7,respectively,all P ＜ 0.05).Conclusion Dectin-1 receptor plays an important role in the phagocytosis of C.albicans by macrophages.
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Objective To determine the expression of interleukin-6 (IL-6) in cystic lesions of patients with acne vulgaris,and to evaluate the in vitro effect of Propionibacterium acnes (P.acnes) on the production of IL-6 and activation of p38 mitogen-activated protein kinase (p38MAPK) in the human acute monocytic leukemia cell line THP-1.Methods Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of IL-6 in cystic lesions of 6 patients with acne vulgaris,as well as in skin tissues of 6 healthy persons.Some cultured THP-1 cells were divided into 5 groups to be treated with 2 × 106 CFU/ml,2 × 107 CFU/ml and 2 × 108 CFU/ml heat-killed P.acnes suspensions (P.acnes groups),100 μμtg/L lipopolysaccharide (LPS group) and RPMI 1640 medium (control group) respectively.After 1-,3-and 6-hour treatment,real-time fluorescence-based quantitative PCR was conducted to determine the mRNA expression of IL-6 in the above groups.Enzyme-linked immunosorbent assay (ELISA) was performed to detect the level of IL-6 in the culture supernatant of cells in the 2 × 108-CFU/ml P.acnes group,LPS group and control group at 24 hours after the treatment.Western blot analysis was conducted to determine the protein expression of p38MAPK and phosphorylated p38MAPK in the 2 × 108-CFU/ml P.acnes group after 15-,30-and 60-minute treatment,as well as in the LPS group after 30-minute treatment and in the control group.Some other THP-1 cells were divided into 3 groups:2 × 108-CFU/ml P.acnes group treated with 2 × 108 CFU/ml P.acnes suspensions,SB203580 (an inhibitor of p38MAPK) group treated with 20 μmol/L SB203580 for 30 minutes followed by the treatment with 2 × 108 CFU/ml P.acnes suspensions,and control group treated with RPMI 1640 medium alone.After 6-hour treatment,the mRNA expression of IL-6 in the above 3 groups was measured by real-time fluorescencebased quantitative PCR.Results The mRNA expression of IL-6 was significantly higher in the cystic lesions of acne vulgaris than in the normal skin tissues (3.680:±:0.790 vs.1.155 ± 0.250,t =3.047,P ＜0.05).Two-way analysis of variance showed that there were significant difference in the mRNA expression of IL-6 among the 2 × 106-CFU/ml,2 × 107-CFU/ml and 2 × 108-CFU/ml p.acnes groups,LPS group and control group (F =532.3,P ＜ 0.001,v =4),and the mRNA expression of IL-6 significantly differed among different time points (F =526.6,P ＜ 0.001,v =2).There were also significant differences in the IL-6 level in the culture supernatant of cells among the 2 × 108-CFU/ml p.acnes group ([1 618.22 ± 32.23] ng/L),LPS group ([3 212.06 ± 353.00] ng/L) and control group ([147.10 ± 0.53] ng/L;v =2,F =102.35,P ＜0.01).After 15-,30-and 60-minute treatment with 2 × 108 CFU/ml P.acnes suspensions,the protein expression of phosphorylated p38MAPK obviously increased.The mRNA expression of IL-6 in THP-1 cells was significantly lower in the SB203580 group than in the 2 × 108-CFU/ml p.acnes group (t =15.91,P =0.004).Conclusions The mRNA expression of IL-6 evidently increases in the cystic lesions of patients with acne vulgaris.P.acnes can activate the signaling molecule p38MAPK in THP-1 cells,and promote the production of IL-6 by THP-1 cells.
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Objective To screen the optimal RNA interference sequence of acute monocytic leukemia associated antigen 22 (MLAA-22) gene in order to study gene function of it. Methods MLAA-22 coding sequence (CDS) was cloned by reverse transcription polymerase chain reaction (RT-PCR) and the CDS was inserted in to pEGFP-N1-3FLAG vector to construct eukaryotic expression vector of MLAA-22. At the same time, four RNA interference sequences were designed and cloned to the vector. Expression vector and RNA interference vector were co-transfected into 293T cells, and the optimal RNA interference sequence was screened by fluorescence and Western blot analyses. Results MLAA-22 eukaryotic expression vector pEGFP-N1-3FLAG and four RNA interference vectors were successfully constructed. After co-transfected 293T cells, KD2 was selected as the optimal interference sequence of MLAA-22. Conclusion KD2 is an optimal interference sequence for targeting MLAA-22 antigen gene.
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Objective To construct the DNA vaccine pIRES2-MLAA34-HSP70,and to detect its immune effect.Methods The acute monocytic leukemia associated antigen gene MLAA-34 and heat-shock protein (HSP)70 gene were extracted by using RT-PCR.The specific overlapping primer was designed,and the fusion gene MLAA34-HSP70 was amplified by using SOE-PCR technique.Then the DNA vaccine pIRES2-MLAA34-HSP70 was constructed,and BALB/c mice were immunized with this DNA vaccine.The splenic lymphocyte killing activity was detected by using MTT,levels of IL-2,IL-4 and IFN-γ were also detected by using ELISA.Results The MLAA34-HSP70 gene (2 956 bp) and the DNA vaccine pIRES2-MLAA34-HSP70 was amplified and constructed successfully.The killing efficiency of DNA vaccine pIRES2-MLAA34-HSP70 in U937 cells was significantly higher than that in other experimental groups and control group (P＜0.01),and levels of IL-4,IL-2 and IFN-γin DNA vaccine pIRES2-MLAA34-HSP70 group were significantly higher than those in the other experimental groups and control group (P＜0.01).Conclnsion The DNA vaccine pIRES2-MLAA34-HSP70 is constructed successfully.It is shown that the DNA vaccine induces strong humoral immunity,which could enhance immune responses to tumor cells and specificlly kill MLAA34 positive cells.
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Objective To investigate the effects of Candida albicans on the expression of tumor necrosis factor-α(TNF-α)and activation of the intracellular signaling molecule p38 mitogen-activated protein kinase(p38MAPK)in a human acute monocytic leukemia cell line THP-1. Methods Some THP-1 cells were divided into several groups in vitro: two C. albicans groups treated with 105 CFU/ml and 106 CFU/ml heat-killed C. albicans respectively, a lipopolysaccharide (LPS)group treated with 100 μg/L LPS, a blank control group treated with RPMI 1640 medium, two dexamethasone-inhibited groups pretreated with 40 μg/L dexamethasone for 30 minutes followed by treatment with 106 CFU/ml heat-killed C. albicans and LPS respectively. After treatment for 1, 3 and 6 hours, real-time fluorescence-based quantitative PCR was performed to measure TNF-α mRNA expression in THP-1 cells in the above groups. Enzyme-linked immunosorbent assay(ELISA)was conducted to determine the level of TNF-α protein in the supernatant of THP-1 cells treated with 106 CFU/ml heat-killed C. albicans, 100 μg/L LPS or RPMI 1640 medium(blank control group)for 24 hours. Western blot was performed to measure the protein expression of p38MAPK and phosphorylated p38MAPK in THP-1 cells after treatment with 106 CFU/ml heat-killed C. albicans or RPMI 1640 medium (blank control group)for 30 and 60 minutes. Statistical analysis was carried out by using two-way analysis of variance, one-way analysis of variance and the least significant difference(LSD)-t test. Results Significant differences were observed in the mRNA expression level of TNF-α among the C. albicans groups, LPS group and blank control group (F = 110.98, P < 0.001). The mRNA expression level of TNF-α in THP-1 cells increased over time in a time-dependent manner after C. albicans treatment, with significant differences among different time points (F = 701.680, P < 0.001). Compared with the blank control group, both 106-CFU/ml C. albicans group and LPS group showed a significant increase in TNF-α protein expression (6385.70 ± 533.99 ng/L and 3212.06 ± 353.00 ng/L vs. 147.10 ± 0.53 ng/L, P < 0.001 and 0.005, respectively). An obvious increase was observed in the expression level of phosphorylated p38MAPK protein, but no significant changes were noted in that of p38MAPK protein, in THP-1 cells treated with 106 CFU/ml C. albicans for 30 and 60 minutes compared with the blank control group. The mRNA expression level of TNF-α significantly decreased in dexamethasone-pretreated 106-CFU/ml C. albicans group and LPS group compared with those without dexamethasone pretreatment(3.77 ± 0.62 vs. 208.50 ± 10.50, 6.20 ± 1.93 vs. 161.35 ± 1.65, both P < 0.001). Conclusions Heat-killed C. albicans can induce the activation of p38MAPK in and secretion of TNF-α by human THP-1 cells, which then participate in the innate immune response against C. albicans.
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Objective To investigate whether 5, 7-dihydrox-8-nitrochrysin (NOC) induces apoptosis in U937 monocytic leukae-mia cells is involved in the regulation of the activity of PKD 2.Methods U937 cell line cells were cultured in vitro .Apoptosis rate was analyzed by flow cytometry (FCM) using propidium iodide (PI) staining.DNA ladder bands were observed by DNA agarose gel electro-phoresis.The phosphorylated protein expression of PKD 2 was analyzed using Western blot .Results NOC (2.5, 5.0, and 10.0μmol/L) increased apoptosis rate in U937 cells in a concentration-dependent manner ( P <0.05).After treatment with NOC (5.0 and 10.0μmol/L) for 24 h, U937 cells presented typical DNA ladder bands .At the same time, not only did NOC effectively down-regulate the ex-pression of PDK2 phosphorylated protein , but also increased apoptosis rate in U 937 cells in the presence of G?6976, a specific inhibitor of PKD2.Conclusions The effect that NOC induces apoptosis in U 937 cells is related to the inhibition of the activity of PKD 2.
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ObjectiveTo evaluate the cytogenetic characteristics of the patients with acute myelomonocytic leukemia (AML-M4), acute monocytic leukemia (AML-M5)and myelodysplastic syndrome (MDS).MethodsChromosomes of bone marrow cells were prepared by short-term culture.Karyotype analysis was performed in 100 AML-M4,46 AML-M5 and 115 MDS by R-banding technique.Results26 ％ (26/100) AML-M4 had clonal cytogenetic abnormalities which mainly include +8,t(8;21) and -7.26 ％ (12/46) AML-M5 had clonal cytogenetic abnormalities which mainly include +11. 39 ％(45/115)had clonal cytogenetic abnormalities which mainly include +8, Hypodiploid and -7. ConclusionCytogenetic detection is very important for the diagnosis of myelodysplastic syndrome and acute myelocytic leukemia.
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Objective To evaluate the effectiveness and side effect of MT regimen (mitoxantrone plus teniposide) in inductive chemotherapy and explore the relationship between the effectiveness and karyotype. Methods 33 patients with acute monocytic leukemia were divided into two groups according to the treatment history or risk status according to cytogenetics MRC criteria. Group A (n=23) and B (n=10) were primary treatment and no remission following one course of DA (daunorubicin plus cytarabine) or HDA (Harringtonine,daunorubicin plus cytarabine) regimen,respectively. According to MRC criteria,group C (n=29) and D (n=4) were intermediate and adverse group. All the cases received two courses MT regimen chemotherapies to induce remission. The results and side effects were analysed. Results The complete remission rate and effective rate in group A and B were 83 % (19/23) and 60 % (6/10),91 % (21/23) and 70 % (7/10) respectively. The complete remission rate and effective rate in group C and D was 83 % (24/29) and 25 % (1/4),88 % (26/29) and 50 % (2/4) respectively. In complex cytogenetic group and 11q23 abnormal without complex cytogenetic group,CR rate was 0 (0/3) and 100 % (4/4). The time point,count of WBC nadir and the duration of WBC were less than 1×109/L is (7±3) day after chemotherapy,(0.4±0.2)×l09/L,(8±5) day. Chemotherapy related mortality was 0. Conclusion MT regimen was highly effective and safe in inducing remission in acute monocytic leukemia,including the cases which achieved no remission following one course of DA or HDA regimen. The effectiveness of MT regimen relates to the cytogenetics. MT regimen may be highly effective in cases with 11q23 abnormal and poor effective in cases with complex cytogenetic.
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Objective To compare the therapeutic and adverse effects of MT regimen (mitoxantrone plus teniposide) and DA regimen (daunorubicin plus cytarabine) on initial treatment to acute monocytic leukemia. Methods 40 patients with initial treatment to acute monocytic leukemia were randomly divided into MT group(n=23) and DA group(n=17). All patients were treated with MT or DA regimen. The result and adverse effects of the two regimens were compared. Results Complete remission(CR) rate in the first course chemotherapy in MT and DA regimen was 65 % and 18 %, respectively. The total CR rate in MT and DA regimen was 83 % and 47 % respectively. The total effective rate was 92 % and 59 %, respectively. Significant differences were found. Severe myelosuppression occurred in both groups. The counts of wbc nadir and the durations of wbc less than 1×10~9/L were not significantly different in two group. The time points of wbc nadir, the start and end time points of wbc less than 1×10~9/L were significantly later in MT group than in DA group. Conclusion MT regimen is significant better than DA regimen in inducing remission in initial treatment acute monocytic leukemia, and it is a good choice for inducing remission strategy. The degrees of myelosuppression in two groups are similar. But the occurrent time of myelosuppression is later in MT group than in DA group. The great attention should be paid to anti-infection and support therapy at time properly.
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Objective To study the effects of antisense oligonucleofide(ASODN)fusion gene MLL-AF9 on cell proliferation and apoptosis of human acute monocytic leukemia cell line THP-1 expressing MLL-AF9.Methods ASODN and sense oligonucleotide(SODN)targeting MLL-AF9 were designed,constructed and transfected into THP-1 by lipofectmine.The level of MLL-AF9 mRNA expression was examined by reverse transcription polymerage chain reaction(RT-PCR)and the expression of MLL-AF9 protein was detected by Western blotting. Cell proliferation rate was analyzed by modified-MTT assay.Hoechst33258 staining Wag used to detect the apoptotic bodies in the cells.The change of apoptosis rate was detected by flow cytometry.Results The level of MLL-AF9 mRNA and protein expression after transfection was significantly decreased in ASODN-treated group in comparison with that in the control group,liposome group and SODN group(P＜0.01).Proliferation of the ASODN-treated cells was inhibited and apoptosis was evidently increased by(10.4±3.0)%,(22.8±2.5)%and(24.7±3.1)% for 0,24 and 48 h,respectively in a time-dependent manner (P＜0.01).Staining of the ASODN-treated cells with Hoechst 33258 showed the morphology characteristic of apoptosis.Conclusion Special ASODN targeting MLL-AF9 effectively inhibited MLL-AF9 expression in THP-1 cell,reduced cell proliferation and induced apoptosis.