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Objective To investigate the effects of shRNA interference (RNAi) targeting the histone lysine specific demethylase 1 (LSD1) on the proliferation and apoptosis in acute leukemia (AL) cells. Methods LSD1 shRNA vectors were constructed and transduced into HL-60 and SHI-1 AL cell lines. Inhibitory efficiency of LSD1 was detected by real-time quantitative PCR (RT-qPCR) and Western blot respectively. Cell proliferation was assessed by methyl thiazolyl tetrazolium (MTT), and cell apoptosis was measured by flow cytometry. Results After interference of LSD1, the expression levels of LSD1 mRNA and protein in HL-60 and SHI-1 cells (mRNA: 0.242 ±0.023, 0.207 ±0.006; Protein: 0.256 ±0.015, 0.486 ±0.042) were decreased compared with blank control group without transfection process (mRNA: 1.021 ±0.178, 1.039 ±0.395; Protein:0.552 ±0.026, 0.754 ±0.060) and empty vector negative control group (shNC group) (mRNA: 0.935 ±0.136, 1.016±0.203;Protein: 0.500±0.026, 0.750±0.049) (P<0.05). The levels of their cell proliferation (absorbance value: 0.712±0.010, 0.549±0.007) were inhibited compared with blank control group (absorbance value:0.823±0.010, 0.625±0.005) and shNC group(absorbance value: 0.818±0.019, 0.621±0.003) (P< 0.05). While cell apoptosis rates were increased [(32.80 ±1.35) %, (23.49 ±1.40) %] compared with blank control group [(8.08 ±0.62) %, (7.28 ±1.17) %] and shNC group [(8.08 ±0.62) %, (7.28 ±1.17) %] (P< 0.05). Conclusions Lentivirus-mediated shRNA interferencing LSD1 can inhibit cells ' proliferation and promote apoptosis of HL-60 and SHI-1 AL cell lines, indicating that LSD1 may be a potential biological molecular marker and a new treatment target for AL.
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Objective To investigate the relationship between the helper T cell 17 (TH 17)/ Regulatory T cells (Treg cells) balance in peripheral blood with acute graft-versus-host reaction (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT),as well as the impact of anti-thymocyte immunoglobulin (ATG) on helper T cells in peripheral blood.Method Seventyeight hematologic patients underwent allo-HSCT,conditioning with or without ATG.Ten healthy volunteers severed as a control group.The helper T and regulatory T cells in peripheral blood were detected by flow cytometry.Enzyme-linked irnmunosorbent assay (ELISA) was used to detect serum concentrations of interleukin(IL)-17,IL-21,IL22,IL23,γ interferon (IFN-γ),and transforming growth factor β1 (TGF-β1).Result The percentage of Treg cells,TH17 cells and ratio of TH17/Treg cells in patients without aGVHD showed no significant difference from the healthy controls (P＞ 0.05).As compared with control group and non aGVHD group,the ratio of Treg cells was increased,the percentage of TH 17 cells,and TH 17/Treg cells were significantly increased in 1-2-degree aGVHD group (P＜0.01).With increased degree of aGVHD,the difference as above was more significant in 3-4-degree aGVHD recipients (P＜0.01).In aGVHD group,the IL-17,IL-23,IL-21 and IFN-γ concentrations were higher than the healthy group (P＜0.01) and non-aGVHD group (P＜0.05).Serum TGF-β1 level in aGVHD group was significantly decreased as compared with healthy group and non-GVHD group (P＜0.05),while IL-22 concentrations showed no statistically significant difference among three groups (P＞0.05).In anti-thymocyte immunoglobulin (ATG) pretreatment group,the absolute count of peripheral blood lymphocytes was less than in healthy control group (P＜0.01).In ATG group,the absolute counts of TH1 cells,TH17 cells,CD3+ CD4+ cells and non-TH1/17 cells were less than in non-ATG group (P =0.0000),while the absolute counts of lymphocytes,CD3+ CD4-cells,and TH 1/17 cells were less than in non-ATG group,but there was significant difference (P＞0.05).Conclusion The balance of TH 17/Treg cells and related cytokines were closely associated with aGVHD after allo-HSCT,and ATG influences the reconstruction of TH 17 and Th1 cells at early stage.
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Objective To explore the impact of rituximab on Th17 cells and related cytokines in patients with diffuse large B-cell lymphoma (DLBCL) in vitro and its significance.Methods 20 cases of DLBCL untreated patients and 20 healthy subjects were enrolled in the name of DLBCL group and health control group, respectively.4 peripheral blood samples were collected from every case to separate peripheral blood mononuclear cells (PBMCs), which were assigned to 4 subgroups according to different culture conditions: blank subgroup(subgroup A), rituximab subgroup (subgroup B), rituximab and serum subgroup (subgroup C) and polarization subgroup (subgroup D) (added IL-6 and TGF-β).After cultured in vitro, the percentage of Th17 cells in each subgroup was tested by flow cytometry, and the cytokine IL-17 in the abovementioned culture fluid was measured by enzyme-linked immunosorbent assay (ELISA).Results In health control group, the percentage of Th17 cells and the level of IL-17 in subgroup D [(17.12 ± 4.90) ％ and (45.735±10.012) pg/ml] were significantly higher than those in subgroup A, B, C (P ＜ 0.05), and there was no difference in each other subgroup A, B, C (P ＞ 0.05).The percentage of Th17 cells and the level of IL-17 in the DLBCL subgroup A were significantly lower than those in health control subgroup A [(0.69±0.24) ％ and (6.012±1.312) pg/ml vs (2.43±0.61) ％ and (8.217±1.681) pg/ml (P ＜ 0.05)].In DLBCL group, after cultured with rituximab, the percentages of Th17 cells in subgroup B, C, D were (2.34±0.48) ％, (2.31±0.53) ％ and (16.92±4.81) ％, and the levels of IL-17 were (7.944±1.538) pg/ml, (7.957±1.533) pg/ml and (44.417±9.881) pg/ml, respectively, which were all significantly higher than those in subgroup A.Besides, the percentage of Th17 cells and the level of IL-17 in DLBCL subgroup D were significantly higher than those in subgroup B, C (P ＜ 0.05), while there was no difference between subgroup B and subgroup C.Conclusion Experiments in vitro confirmed that the percentage of Th17 cells in PBMCs of DLBCL patients was lower than that in healthy persons, and rituximab could elevate the percentage of Th17 cells in PBMCs of DLBCL patients.
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Objective To explore the effect of recombinant human granulocyte colony stimulating factor (rhG-CSF) mobilization on TH17/Treg cells and its impact on suppressor of cytokine signaling-3 (SOCS3) gene expression in CD4+ T cells in donors' peripheral blood.Method Sixteen donors were injected subcutaneously with rhG-CSF 5 μg/kg every day for 5 consecutive days for peripheral blood stem cells mobilization.At the first 0,3,5 day,the mononuclear cclls (MNCs) in peripheral blood or graft and serum specimens were taken.The CD4 + T cells in MNCs were sorted using immuno-magnetic beads.The ratio of TH 17 and Treg cells in MNCs,cytokines concentrations of IL-17A,IL-21,ID23 and TGFβ1 in serum,and SODC3 gene expression in CD4+ T cells were detected by using flow cytometry,ELISA,and reverse transcription real-time quantitative PCR (RT-qPCR),respectively.Results (1)The ratio of Th17 cells (CD3+ CD8 CD17+) and Treg cells (CD4+ CD25+ Foxp3+) in MNCs in peripheral blood and graft at the first 0,3 and 5 days after mobilization was (2.69 ± 0.81) ％,(0.91 ± 0.33) ％,(0.35 ± 0.12) ％,(0.21 ± 0.05) ％,and (0.56 ± 0.24) ％,(0.72 ± 0.22％),(1.59 ± 0.54) ％,(3.38 ± 0.52) ％,respectively,showing a significant declining and increasing trend respectively (P＜0.05); (2)The cytokine concentrations in serum at the first 0,3 and 5 days after mobilization were 7.33 ± 0.89,5.78 ± 1.03 and 3.32 ± 0.84 μg/L for IL-17A; 124.56 ± 15.18,117.12 ± 14.45 and 64.94 ± 11.25 μg/L for IL-21 ; 183.52 ± 59.35,280.49 ± 69.75 and 393.62 ± 57.25μg/L for TGF-β1 (P＜0.01) ; and 45.89 ± 6.95,46.25 ± 7.44 and 47.45 ± 10.75 μg/L for IL-23,respectively.The IL-17A and IL-21 concentrations showed significant declining trend,contrarily TGF-β1 with an increasing trend,while IL-23 concentration had no change.After rhG-CSF mobilization,the SOCS3 gene expression in CD4 + T cells of peripheral blood and graft at the first 0,3,5 days was gradually increased.Conclusion rhG-CSF suppresses Th17 cells and promotes regulatory T cells generation,meanwhile decreases IL-17A and IL-21 and elevates serum TGF-β1 concentrations,and contributes to CD4 + T cells differentiation to Tregs,probably by elevating SOSC3 gene expression in CD4+ T cells.
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Objective To understand the changes of Th17 cells and related cytokines in diffuse large B-cell lymphoma (DLBCL) patients treated with rituximab and its significances.Methods Patients were assigned to 4 groups,there were 20 cases in the control group,31 cases in the initial treatment group,31 cases in CHOP group and 25 cases in RCHOP group.The percentage of Th17 cells in the peripheral blood of each group was tested by flow cytometry,the related cytokines IL-17,IL-21,IL-23,TGF-β in the peripheral blood were measured by enzyme-linked immunosorbent assay.Results The percentage of Th17 cells and the levels of IL-17,IL-21,IL-23 in the initial treatment group [(0.67±0.21) ％,(5.929±1.342) pg/ml,(130.632±17.945)pg/ml,(51.681±9.808) pg/ml] and the CHOP-CR group [(1.07±0.37) ％,(6.526±0.538) pg/ml,(132.119±7.700)pg/ml,(50.245±7.668) pg/ml] were both significantly lower than those in the control group[(2.53±0.63) ％,(8.435±2.031) pg/ml,(149.265±12.316) pg/ml,(55.303±7.778) pg/ml] (P ＜ 0.05).The level of TGF-β in the initial treatment group [(370.615±98.444) pg/ml] was significantly higher than that in the control group [(311.895±73.365) pg/ml] (P ＜ 0.05).The percentage of Th17 cells and the levels of IL-17,IL-21,IL-23 in the RCHOP-CR group [(2.38±0.59) ％,(7.724±0.780) pg/ml,(148.412±7.355) pg/ml,(55.668±7.532) pg/ml] were significantly higher than those in the initial treatment group [(0.67±0.21) ％,(5.929±1.342) pg/ml,(130.632±17.945) pg/ml,(51.681±9.808) pg/ml] and the CHOP-CR group [(1.07±0.37) ％,(6.526±0.538) pg/ml,(132.119±7.700) pg/ml,(50.245±7.668) pg/ml] (P ＜ 0.05).The level of TGF-β in the RCHOP-CR group[(283.904±59.223) pg/ml] was significantly lower than that in the CHOP-CR group [(341.481±95.597) pg/ml] (P ＜ 0.05).Conclusion Th17 cells might be negatively correlated with the DLBCL development,the reduced IL-23 and elevated TGF-β might suppress the differentiation of Th17 cells.Rituximab could elevate the percentage of Th17 cells in DLBCL patients,and it is related with the effect of chemotherapy.
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Objective To explore clinical features of chemotherapy-induced graft-versus-host disease and to observe the dynamical changes of Th17/Treg ratio and related cytokines in one relapsed acute T-lymphoblastic leukemia after allo HSCT.Methods Twenty health volunteers were healthy controls.One patient achieved complete haematologic remission after two courses of chemotherapy and underwent matched HLA identical sibling allogenic peripheral blood stem cell transplantation,donor cell implanted state,disease recurrence and graft-versus-host disease were observed and Th17/Treg cells and its related factors in the peripheral blood were detected in different periods dynamically changes by methods of flow cytometry and ELISA.Results The patient achieved complete donor chimerism (FDC) at day +27 after transplantation.Hematopoietic function was fully recovered at day +34.Chronic GVHD (cGVHD) occurred at day +110.Thereafter,extramedullary relapse occurred in cGVHD state at day +264.After one course of Hyper-CVAD chemotherapy,patient complicated overlap syndrome with cGVHD of oral cavity ulcer and degree Ⅳ intestinal aGVHD at day +294 and day +347,respectively.Th17 cell ratio in CD+4 T cells (1.7 ％) in the overlap syndrome slightly increased,compared with control group [(0.56±0.35) ％].The ratio of Treg cells in CD+4 T cells (4.66 ％) with cGVHD increased compared with normal control group [(0.59±0.33) ％],recurrence (0.39 ％),and hematopoietic stem cell implantation (1.15 ％),but the ratio of Treg cells increased significantly when patient complicated overlap syndrome (7.83 ％).The ratios of Th17/Treg were more than 1 at relapse stage and less than 1 at GVHD stage.The IL-17A level in serum significantly increased in cGVHD (6.114 pg/ml)and overlap syndrome (6.805 pg/ml) stage compared with normal control group [(5.19±0.77) pg/ml].TGF-β1 levels were significantly higher at different periods after transplantation compared with control group [(48.81±4.9) ng/ml].Conclusion Chemotherapy can induce GVHD in relapsed acute leukemia patients after hematopoietic stem cell transplantation,and the imbalance of the Th17/Treg cells and its cytokines maybe related with disease relapse and GVHD.
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Objective To understand the changes of allergic rhinitis from immunology,pathophysiology and morphology method were applied.Methods Toluene 2,4-diisocyanate was dissolved with florence oil to final concentration 10%,this solution was dropped into the nasal vestibules of animals to induce sensitization process.8 guinea pigs were prepared as allergic rhinitis with 10% Toluene 2,4-diisocyanate,and other 8 animals were used as blank controh.After model successfully established,the blood were obtained to determine RBC-C3b receptor rosette rate and RBC-imuuunocomplex rosette rate.The nasal mucosas were obtained from 2 groups,distribution and changes of substance P in nasal mucosa were observed with the application of immunohistochemical staining.The content of blood histamine was determined with ELASA method.The pathological changes of nasal mucosas were observed with the application of light microscope and transmission electron microscope.Results The behavior scores of the modeling animals were significantly higher than that of controls(P＜0.01).The value of RBC-C3b reeeptor rosette rate and RBC-imuuunoeomplex rosette rate of the modeling animals were significantly decreased than that of the controls(P＜0.05).Substance P presented in the normal nasal mucosal epithelial cells,epithelium cells of blood vessels,glandular cells and its duets,the staining density and the positive staining cells in the same aero in modeling group significantly increased,compared with controls.The counts of substance P-positive cells of control group were less than those of modeling group(P＜0.05).The content of blood histamine of the modeling animals were significantly increased compared with the controls(P＜0.01).The structure of false multiple coat cilium columnar epithelial cells in nasal mucosa of control group were successive,intact,and distinct.There were normal mucosal epithelium,lamina propria and submucosa.But modeling group showed that mucosal epithalamiums were damaged and shed,goblet cell proliferated,squamous metaphase,and epithelial necrosis happened,serous glands in lamina propria vigorously proliferated,blood vessels expanded,tissue edema formed,plenty of inflammatory cell such as eosinophil and mast cells were more in number and infiltrated.The structure of epithelial eells,cilia and mierotubule of nasal mucosa of control group were regular and distinct,there were abundant cellular organs in cytoplasm.Eosinophil cells were intact.But iu model group,mucosal epithalamium,cilia and its microtubule,mierovillus,goblet cell were damaged,the cell bulk and nucleus changed,mast cells and eosinophil cells changed,blood vessels expanded,serous glands vigorously proliferated.This morphological change was roughly identical to clinical manifestation of allergic rhinitis.Conclusion Toluene-2,4-diisocyanate can be used to establish allergic rhinitis model in guinea pigs,and some ehanges of the allergic rhinitis in model guinea pig were similar with clinic observation.
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Objective To establish a method of testing lysophosphatidic acid (LPA) by enzymatic cycling. Methods The detection system was developed on the principle of enzymatic cycling method and characteristic of LPA. The methodology was evaluated and the content of LPA was tested in 20 samples of plasma with ovarian carcinoma. Results The linearity was 18.6?mol/L and the detection limit was 0.03?mol/L. The within-run and between-run coefficient of variations were 5.3% and 7.4%, respectively. The recovery was 100.7% and there was a significant correlation with the liquid chromatography method(Y=0.953X+0.51). The reagent system was stable and different concentrations of bilirubin, hemoglobin, triglyceride, glucose, urea and ascorbate did not affect the measurement for the assay. The content of LPA in 20 samples of plasma with ovarian carcinoma was significantly higher than that in control group (P
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Objective To evaluate the efficacy and complications of nonmyeloablative stem cell transplantation(NST) for the treatment of nonmalignant haematologic diseases with high-risk rejection.Methods NST was performed for two patients with severe aplastic anemia(SAA) and one with beta-thalassemia major(TM).The protocal was designed on transplantation of granulocyte colony-stimulating factor(G-CSF) primed allogeneic bone marrow cells combined with perpheral blood stem cells(PBSCs) for two SAA patients,with conditioning regimen based on anti-lymphocyte globulin and reduced dose of cyclophosphamide(CTX).One TM patient was performed transplantation of PBSCs with conditioning regimen of anti-T-lymphocyte globulin(ATG),fludarabine and reduced dose of busulfan.Cyclosporin A combined with methylprednisone was used for graft-versus-host disease(GVHD) prophylaxis.Donor stem cells infusion(DSI) were underwent for three patients at 78,99 and 44 days post transplant respectively.Results Three patients achieved engraftment successfully with mixed chimera and the lowest white blood cell(WBC) of 0.26?10~9/L,0.5?10~9/L and 1.26?10~9/L respectively.The absolute neutrophil count achieved more than 0.5?10~9/L and platelet count achieved more than 20?10~9/L at days of 12d,3d,0d and 1d,5d,0d post transplant in three patients,respectively.The haematopoiesis and chimera were improved after DSI without complications of infection and GVHD in three patients.Conclusion The stem cells engraftment is achieved successfully with donor stem cell infusion followed NST for the treatment of nonmalignant haematologic disease patients with high-risk rejection.