ABSTRACT
[ABSTRACT]OBJECTIVETo explore the occurrence and development of nasal polyps by studying the interleukin 17 and vascular endothelial growth factor expression level in nasal polyp tissues.METHODSNasal polyps in 30 patients with chronic sinusitis (with polyps), ethmoid sinus mucosa in 30 patients with chronic sinusitis (without polyps), inferior turbinate mucosa in 10 patients with nasal septum deviation were collected intraoperatively. IL-17 and VEGF expression were detected using immunohistochemistry SP testing, and correlation between them was analysed. RESULTS1. The expression of IL-17 and VEGF in nasal polyps group is significantly higher than that of the other two groups.There was significant difference between each two groups statistically (P<0.01). 2. The expression of IL-17 was positively correlated with VEGF expression in nasal polyps.CONCLUSION1. The expression of IL-17 and VEGF increased in nasal polyp tissues. 2. The expression of IL-17 and VEGFwas positively correlated in nasal polyps , and both of them take part in the occurrence and development of nasal polyps.
ABSTRACT
Leukemia seems to depend on a small population of "leukemia stem cells (LSCs)" for its growth and metastasis. However, the precise surviving mechanisms of LSCs remain obscure. Cellular senescence is an important obstacle for production and surviving of tumor cells. In this study we investigated the activated state of a pathway, in which reactive oxygen species (ROS) induces cellular senescence through DNA damage and phophorylation of p38 MAPK (p38), in myeloid leukemic CD34(+)CD38(-) cells. Bone marrow samples were obtained from patients with acute myeloid leukemia (AML, n=11) and chronic myeloid leukemia (CML, n=9). CD34(+)CD38(-) cells were isolated from mononuclear cells from these bone marrow samples, and K562 and KG1a cells (two kinds of myeloid leukemia cell lines) by mini-magnetic activated cell sorting. Hematopoietic stem cells (HSCs) from human cord blood served as controls. Intracellular ROS level was detected by flow cytometry. DNA damage defined as the γH2AX level was measured by immunofluorescence staining. Real-time RT-PCR was used to detect the expression of p21, a senescence-associated gene. Western blotting and immunofluorescence staining were employed to determine the p38 expression and activation. The proliferation and apoptosis of CD34(+)CD38(-) cells were detected by MTT assay and flow cytometry. Our results showed that ROS and DNA damage were substantially accumulated and p38 was less phosphorated in myeloid leukemic CD34(+)CD38(-) cells as compared with HSCs and H(2)O(2)-induced senescent HSCs. Furthermore, over-phosphorylation of p38 by anisomycin, a selective activator of p38, induced both the senescence-like growth arrest and apoptosis of CD34(+)CD38(-) cells from K562 and KG1a cell lines. These findings suggested that, although excessive accumulation of oxidative DNA damage was present in LSCs, the relatively decreased phosphorylation of p38 might help leukemic cells escape senescence and apoptosis.
Subject(s)
Female , Humans , Male , ADP-ribosyl Cyclase 1 , Metabolism , Antigens, CD34 , Metabolism , Cellular Senescence , Leukemia, Myeloid, Acute , Pathology , Neoplastic Stem Cells , Metabolism , Pathology , Oxidative Stress , Phosphorylation , Reactive Oxygen Species , Metabolism , Tumor Cells, Cultured , p38 Mitogen-Activated Protein Kinases , MetabolismABSTRACT
Leukemia seems to depend on a small population of "leukemia stem cells (LSCs)" for its growth and metastasis. However, the precise surviving mechanisms of LSCs remain obscure. Cellular senescence is an important obstacle for production and surviving of tumor cells. In this study we investigated the activated state of a pathway, in which reactive oxygen species (ROS) induces cellular senescence through DNA damage and phophorylation of p38 MAPK (p38), in myeloid leukemic CD34(+)CD38(-) cells. Bone marrow samples were obtained from patients with acute myeloid leukemia (AML, n=11) and chronic myeloid leukemia (CML, n=9). CD34(+)CD38(-) cells were isolated from mononuclear cells from these bone marrow samples, and K562 and KG1a cells (two kinds of myeloid leukemia cell lines) by mini-magnetic activated cell sorting. Hematopoietic stem cells (HSCs) from human cord blood served as controls. Intracellular ROS level was detected by flow cytometry. DNA damage defined as the γH2AX level was measured by immunofluorescence staining. Real-time RT-PCR was used to detect the expression of p21, a senescence-associated gene. Western blotting and immunofluorescence staining were employed to determine the p38 expression and activation. The proliferation and apoptosis of CD34(+)CD38(-) cells were detected by MTT assay and flow cytometry. Our results showed that ROS and DNA damage were substantially accumulated and p38 was less phosphorated in myeloid leukemic CD34(+)CD38(-) cells as compared with HSCs and H(2)O(2)-induced senescent HSCs. Furthermore, over-phosphorylation of p38 by anisomycin, a selective activator of p38, induced both the senescence-like growth arrest and apoptosis of CD34(+)CD38(-) cells from K562 and KG1a cell lines. These findings suggested that, although excessive accumulation of oxidative DNA damage was present in LSCs, the relatively decreased phosphorylation of p38 might help leukemic cells escape senescence and apoptosis.
ABSTRACT
This study investigated the correlation between and compared the effects of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase α (p38MAPKα) in the ex vivo expanded umbilical cord blood (hUCB) CD133(+) cells. hUCB CD133(+) cells were cultured in the hematopoietic stem cells (HSCs) culture medium with N-acetylcysteine (NAC, an anti-oxidant), p38MAPKα-specific inhibitor (SB203580) or their combination. The levels of ROS and expression of phosphorylated p38MAPKα (p-p38) in CD133(+) cells were flow cytometrically detected. The efficacy of ex vivo expansion was evaluated by the density of CD133(+) cell sub-group colony-forming cells (CFC) and cobblestone area-forming cells (CAFC) assay. Our results showed decreased ROS levels in NAC, SB203580, and their combination treatment groups were almost 37%, 48%, and 85%, respectively. Furthermore, SB203580 abrogated the activation of p38MAPKα more obviously than NAC. Moreover, the CD133(+) cells in SB203580 treatment group had a 21.93±1.36-fold increase, and 14.50±1.19-fold increase in NAC treatment group, but only 10.13±0.57-fold increase in control group. In addition, SB203580 treatment led a higher level increase in the number of CFU and CAFC than NAC did. These findings suggested that, in expanded CD133(+) cells, ROS activates p38MAPKα, which, in turn, induces ROS production, and p38MAPKα might be the most suitable regulator in ROS-p38MAPKα pathway for the promotion of HSCs ex vivo expansion.
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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.
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OBJECTIVE To investigate the characteristic computer tomography (CT) changes in cases of one-sided chronic ethmoid-maxillary sinusitis. METHODS The CT results of 76 patients diagnosed with one-sided chronic ethmoid-maxillary sinusitis were reviewed. RESULTS There were 51 patients with one-sided chronic ethmoid-maxillary sinusitis on the right side and 25 patients on the left side. There were 48 cases of obstruction of the ostium of the maxillary sinus. The characteristics of soft tissue lesions were mucosa hypertrophy and polypiform density spot. There were 9 cases with bony destruction. CONCLUSION More cases with pathologic changes on the left side were found. The obstruction of the ostium of the maxillary sinus and the abnormal anatomy of ostiomeatal complex were identified as important anatomical features of one-sided chronic ethmoid-maxillary sinusitis.
ABSTRACT
In order to explore a new special and effective way to prevent graft versus host disease (GVHD) after allogenic bone marrow transplantation (allo-BMT), the stem cell antigen-1 (Sca-1) + early hematopoietic cells (EHC) from BALB/c mouse (H-2d) were introduced with exogenous mouse Fas ligand (mFasL) cDNA gene by the retrovirus-mediated gene transfer and expanded for one week, and then they were co-cultured with the spleen mononuclear cells (SMNC) from BAC mouse (H-2dxb) as one way mixed lymphocyte reaction (OWMLR). The cytotoxicity of treated BAC mouse SMNC against Na2 51CrO4 labeling SMNC from BALB/c mouse was observed. The bone marrow mononuclear cells (BMMNC) from BAC mouse treated by the above methods were transplanted into lethally-irradiated congenic BALB/c mice to observe the occurrence of GVHD. The results showed that the SMNC from BAC mouse after OWMLR with exogenous mFasL cDNA gene-transduced hematopoietic cells (HC) from BALB/c mouse in a ratio of 1 to 5 exhibited an obvious inhibition of the cytotoxicity against the BALB/c mouse spleen cells at different effector/target ratios as compared to the control group (P<0.01). The grade I GVHD or no GVHD and the 80% survival rate at day 60 post-BMT were observed in the BALB/c mouse receiving BAC mouse BMMNC treated with similar way, while the grade II - III GVHD and the 20% survival rate were noted in the control group (P<0.01). It is suggested that the attenuation of GVHD in allo-BMT recipient could be successfully achieved through FasL-Fas pathway in an H-2 haplotype disparate mouse combination.
Subject(s)
Animals , Female , Mice , Rats , Bone Marrow Transplantation , Fas Ligand Protein , Graft vs Host Disease , Allergy and Immunology , Therapeutics , H-2 Antigens , Genetics , Haplotypes , Hematopoietic Stem Cells , Cell Biology , Allergy and Immunology , Membrane Glycoproteins , Allergy and Immunology , Mice, Inbred BALB C , Mice, Inbred C57BL , Rats, Wistar , Signal Transduction , Spleen , Cell Biology , Allergy and Immunology , T-Lymphocytes , Allergy and Immunology , Transfection , fas Receptor , Allergy and ImmunologyABSTRACT
To investigate the value of apoptosis of the allo-antigen specific T cells induced by Fas/FasL pathway in preventing graft-versus-host disease (GVHD), the CD34+ cells transfected with FasL or not, used as stimulus cells, were mixed with allo-antigen specific T lymphocytes in presence or absence of IFN-gamma and IL-2. After 5 days, apoptosis of T cells was detected by TdT nick end mediated dUTP labeling (TUNEL) and flow cytometry (FCM). The affects of these two cytokines on CD34+ cells in the graft were also compared. The ratio of apoptosis of T cells was 12.1+/-1.5% when CD34+ cells transfected with FasL was used as stimulus cells, much higher than that of CD34+ cells non-transfected (3.2+/-1.1%, P<0.01). And in presence of IFN-gamma or IL-2, the ratio reached 20.1+/-2.3%, 17.6+/-1.3% respectively (P<0.01). However, IFN-gamma up-regulated Fas expression of CD34+ cells and increased the sensibility of CD34+ cells to soluble FasL (sFasL); IL-2 showed no such effect. It is possible to induce apoptosis of the allo-antigen specific T cells of grafts activated by allo-antigen by exogenous Fas ligand expressed on recipient cells and this might provide a new approach for preventing GVHD and IL-2 may be more suitable for clinical application.
Subject(s)
Antigens, CD34 , Allergy and Immunology , Apoptosis , Cytotoxicity, Immunologic , DNA, Complementary , Genetics , Fas Ligand Protein , Graft vs Host Disease , Interferon-gamma , Allergy and Immunology , Interleukin-2 , Allergy and Immunology , Membrane Glycoproteins , Allergy and Immunology , T-Lymphocytes , Cell Biology , Physiology , fas Receptor , Allergy and ImmunologyABSTRACT
In order to regulate the apoptosis induced by Fas-FasL system, a soluble isoform of mouse Fas was cloned from thymocytes of immature mice with the primers designed according to the full-length Fas cDNA sequence in the GeneBank. It was directionally inserted into the intermedium vector pUC19. DNA sequencing proved that it was consistent with the expected sequence. Then it was subcloned into the eukaryotic expression vector pCA13, which was used to construct the recombinant vector pCA13-FasC. By lipofectamine (LF2000)-mediated transfection, pCA13-FasC was transfected into the 293 cells. RT-PCR and Western blot indicated that the murine soluble Fas C protein was expressed in the 293 cells. Apoptosis inducing test showed that the expression of this murine Fas C could block the Fas-induced apoptosis, which confirmed the biological activity of the recombinant Fas C.
Subject(s)
Animals , Mice , Amino Acid Sequence , Animals, Newborn , Base Sequence , Cloning, Molecular , DNA, Complementary , Genetics , Fas Ligand Protein , Gene Expression , Membrane Glycoproteins , Genetics , Molecular Sequence Data , Recombinant Proteins , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transfection , fas Receptor , GeneticsABSTRACT
To assess the value of CD34+ cells transferred exogenous Fas ligand (FasL) in inducing apoptosis of human leukemic cells, the CD34+ cells transfected with FasL or without, pretreated with mitomycin C, was mixed with leukemic cell line U937 cells in presence or absence of daunorubicin (DNR) or cytosine arabinoside (Ara-C). After 18 h, apoptosis of cells was detected by FCM and TUNEL. Induced for 18 h by CD34+ cells transfected with FasL or without, the ratio of apoptosis of U937 cells was (5.0 +/- 1.3)%, (10.8 +/- 0.6)% (P < 0.01), respectively. Induced by FasL+ CD34+ + DNR, FasL+ CD34+ + Ara-C, the ratio was (13.4 +/- 1.0)% (P < 0.05), (17.9 +/- 1.3)% (P < 0.01), respectively. The result demonstrated that CD34+ cells transfected with exogenous FasL could induce apoptosis of human leukemic cells and showed a cytotoxic synergistic effect when used in combination with chemotherapeutic drugs, suggesting that it was possible to develop a new method in treatment of leukemia.
Subject(s)
Humans , Antigens, CD34 , Apoptosis , Cell Communication , Physiology , Cytarabine , Pharmacology , DNA, Complementary , Genetics , Daunorubicin , Pharmacology , Fas Ligand Protein , Membrane Glycoproteins , Genetics , Mitomycin , Pharmacology , Transfection , U937 Cells , fas Receptor , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To explore a new method of alleviating graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT) through selected elimination of mouse alloreactive T cells (ARTC) by Fas-Fas ligand (FasL) passway.</p><p><b>METHODS</b>The Sca-1(+) early hematopoietic cells (EHCs) were isolated from BALB/c mouse (H-2(d)) bone marrow mononuclear cells (BMMC) by using a high gradient magnetic cell sorting system (MACS), then transferred with exogenous mouse FasL (mFasL) gene by retroviral gene transfecting technique. Afterward the transduced EHCs were expanded in vitro for one week followed by coculture with the spleen cells from BAC mouse (H-2(d) x b) as one-way mixed lymphocyte culture (OWMLC) for 6 days, then the cytotoxicity of treated BAC mouse spleen cells against Na(2)(51)CrO(4) labelling spleen cells from BALB/c mouse was observed.</p><p><b>RESULTS</b>The Sca-1(+) EHCs were successfully isolated by MACS, with a purity of (89.0 +/- 6.1)%. After transferred with exogenous mFasL gene and expanded for one week, the transferred EHCs in the 6 day OWMLC with the spleen cells from BAC mouse at a ratio of five to one resulted in an obvious inhibition of the BAC mouse spleen cells cytotoxicity against the BALB/c mouse spleen cell at different effector/target ratios as compared to the control group (P < 0.01).</p><p><b>CONCLUSION</b>The higher exogenous mFasL-expressing mouse EHCs can deplete ARTC against their own major histocompatibility complex (MHC) antigens in vitro.</p>
Subject(s)
Animals , Female , Mice , Antigens, Ly , Allergy and Immunology , Fas Ligand Protein , Graft vs Host Disease , Allergy and Immunology , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells , Cell Biology , Allergy and Immunology , Membrane Glycoproteins , Genetics , Allergy and Immunology , Membrane Proteins , Allergy and Immunology , Mice, Inbred BALB C , Mice, Inbred C57BL , Signal Transduction , Spleen , Cell Biology , Allergy and Immunology , T-Lymphocytes , Allergy and Immunology , TransfectionABSTRACT
Objective To explore the new method to prevent graft versus host disease (GVHD) by clearing T lymphocytes of bone marrow graft through Fas-FasL way. Methods FasL-cDNA was transfected into BALB/C mouse hematopoietic cells by liposomes. The transfected cells were cultured together with BAC mice bone marrow graft. The mixed bone marrow graft was injected into BALB/C mouse recipients after 60 Co-? ray irradiating. Then the mortality, manifestation and pathologic change of GVHD of recipient mice were observed. Results Compared with control group, the mortality of the recipients in the experimental group was markedly decreased in 60 days (20% vs 70%, P