BACKGROUND:It is discovered in our preliminary study that the activity of psoriatic bone marrow hematopoietic cells is abnormal.OBJECTIVE:To reveal level of stem cell factor(SCF) and granulocyte colony-stimulating factor(G-CSF) secreted by bone marrow stromal cells in psoriasis patients.DESIGN,TIME AND SETTING:This case-control observation experiment was performed at the Laboratory of Department of Dermatology,Taiyuan Central Hospital from October 2007 to August 2008.PARTICIPANTS:Twenty-four common-psoriasis outpatients were selected at the Department of Dermatology,Taiyuan Central Hospital,including 16 males and 8 females,aged 16-59 years old.Twenty controls were selected from Department of Hematology,Taiyuan Central Hospital,whose age and gender were matched with psoriasis patients.METHODS:Bone marrow mononuclear cells were isolated from psoriasis patients and controls using the density gradient centrifugation.Bone marrow stromal cells were cultured by the adherent method.When the cells were cultured for three passages and 72 hours,bone marrow stromal cells and supernatant liquid were collected.MAIN OUTCOME MEASURES:The phenotypes of cells were identified by flow cytometry and the level of SCF and G-CSF were detected by enzyme linked immunosorbent assay kit(ELISA).RESULTS:Flow cytometry assay showed that over 90% bone marrow stromal cells were positive for CD29,but negative for CD34,CD45 and HLA-DR.That is,the purity of bone marrow stromal cells was over 90%.The levels of SCF and G-CSF in psoriasis patients were significantly higher than that in controls(P

Objective To study the levels of soluble tumor necrosis factor receptor (sTNFR)、 tumor necrosis factor alpha(TNF? ) in systemic lupus erythematosus(SLE) patients and its clinical significance. Methods Double antibody sandwich ELISA was used to determine soluble tumor necrosis factor receptor typeⅠ (sTNFRⅠ )、 soluble tumor necrosis factor receptor typeⅡ (sTNFRⅡ ) and TNF? levels in patient serum and peripheral blood mononuclear cell(PBMC) culture supernatants. Other immunological tests were carried out simultaneously. Results① The serum sTNFRⅠ、 sTNFRⅡ、 TNF? sTNFRⅠ /TNF? and sTNFRⅡ /TNF? ratios were significantly higher in SLE patients than those in normal control group(P

Objective:To study methods for inducing CD34+ cells of human bone marrow to differentiate into T cells in vitro to provide theory and method basics for the investigation of activity of T cells derived from psoriatic bone marrow CD34+ cells and establish a technological platform to investigate T lymphopoiesis activity of hematopoietic cells.Methods:Bone marrow CD34+ hematopoietic progenitor cells were isolated by immunomagnetic cell selection and induced differentiate into T cells in the bone in the marrow and thymic stromal microenvironment.Immunfluorescence dying method and flow cytometry analysis were performed to detect CD1-CD3+ cells,CD3+CD4+CD8-cells and CD3+CD4-CD8+ cells dynamically.Results:In the first week,the non-adhension cells were composed mostly of immature CD1+CD3-cells and CD1+CD3+ cells and small proportion of mature CD1-CD3+ cells.In the following analysis,the proportion of immature cells rapidly decreased and CD1-CD3+ cells increased.After 1 week culture,CD4+CD8+ double positive T cells and a small population CD4+CD8-and CD4-CD8+ could be detected among the CD3+ cells.In the following culture,the proportion of CD4+CD8+ double positive T cells decreased significantly and single positive T cells increased gradually.However,small proportion of mature T cells could be detected in the early stage and cann't be found after 4 weeks in the culture system without thymic stromal cells.Conclusion:Mature single positive T cells can develop from CD34+ hematopoietic progenitor cells in the bone marrow and thymic stromal microenvironment and the thymic stromal cells are vital for T lymphopoiesis.

Objective:To investigate the influence of histamine on intedeukin(IL-2) production and proliferation of CD4+ T lymphocytes and CD8+ T lymphocytes.Methods:Density gradient centrifugalization and absorption technique were respecitively applied to detach peripheral blood mononuclear cells(PBMC) and peripheral blood lymphocytes(PBLC). CD4+ and CD8+ T lymphocytes divided with anti-CD4+ and anti-CD8+ antibody were cultured in vitro. IL-2 levels in supernatant were quantified by enzyme-linked immunosobent assay (ELBA), and proliferation index were measured by MTT methods. Results:IL-2 levels in supernatant and proliferation rate of CD4+ and CD8+ T lymphocytes treated with histamine were significantly lower than those of their corresponding un-stimulated controls(P

Objective:To compare the seeding efficiency and graft versus host disease (GVHD) of severe combined immunodeficient (SCID) mice transplanted with human hematopoietic cells intraperitoneally (ip) and intravenously (iv) and to explore the method to set up psoriatic animal model by xenogeneic bone marrow transplantation.Methods:Normal human bone marrow mononuclear cells (BMMNC) were separated by density gradient centrifugation and BMMNC (4?10~7) were injected into lethally irradiated SCID mice by ip or iv injection. GVHD symptom and periphery blood white blood cell resuming dynamics in mice were observed after xenotransplantation and flow cytometry was performed to detect human source CD45~+ cells proportion in periphery blood and bone marrow of mice.Results:The mice transplanted by tail intravenous injection presented obvious GVHD symptoms promptly 2 weeks after transplanting and only one mouse survived in 12 weeks. Among the mice received tail intravenous injection and dealed with cydosporin(CsA) and methotrexate(MTX),some of the mice showed slight GVHD symptom and survival rate was 80%(8/10) in 12 weeks. Slight GVHD symptoms appeared after human bone marrow transplantation by intraperitoneal injection and then most of mice returned to the normal and the survival rate was 70%(7/10) in 12 weeks. The periphery blood white blood cells resuming dynamics, CD45~+ cell proportion of periphery blood and bone marrow after transplantation show no significant difference between the groups transplanted by intravenous and intraperitoneal injection.Conclusion:Human hematopoietic cells could home to bone marrow in SCID mice and result in hematopoietic reconstitution. The transplantation method by intraperitoneal injection, which showed efficient seeding capability, can be used to both bone-marrow transplantation and reducing GVHD.

Objective To study the effects of TNF -?and sTNFRⅠon IL -8production and proliferatio n of psoriatic keratinocytes.Methods In vitro cultured keratinocytes were treated with TNF -?,sTNFRⅠand an-ti -TNFRⅠ.IL -8levels in supernatants were me asured by enzyme-linked immunosobent assay.Expression of IL -8mRNA in keratinocytes was observed by semiquantitive RT -PCR and Southern blotting.Proliferation index wa s measured by MTT colorimetry.Results IL -8levels,mRNA expression and pro liferation index were significantl y higher in psoriatic patients than th ose in control group(P

Objective To study the influence of culture supernatant of psoriatic peripheral blood mononuclear cells (PBMCs) on the colony-forming of bone marrow-derived hematopoietic stem cells and progenitor cells. Methods Bone marrow-derived mononuclear cells were separated by density gradient centrifugation. Methylcellulose semi-solid culture medium was used to culture the bone marrow mononuclear cells in culture systems. The PBMC culture supernatant from psoriatic patients or normal controls were added to the culture, to observe their influence on the marrow-derived high proliferative potential colony forming cells (HPP-CFCs), erythroid and granulocyte-macrophage colony forming units (CFUs-E and CFUs-GM) of bone marrow hematopoietic stem/progenitor cells from healthy individuals. Results The values of HPP-CFCs, CFUs-E and CFUs-GM were significantly lower in the bone marrow cells stimulated by the supernatant of cultured psoriatic PBMCs than those stimulated by the supernatant of cultured normal PBMCs and those in the spontaneous proliferation group (P 0.05). Conclusions Psoriatic PBMCs have specific biological activity and can inhibit the colony forming of bone marrow hematopoietic cells from healthy individuals.

Objective To study the in vitro activity of T cells differentiated from bone marrow CD34+ cells of psoriatic patients with family histories. Methods Bone marrow CD34+ cells were isolated from psoriatic patients with family history and normal persons by immunomagnetic cell selection and induced to differentiate to T cells in thymic stromal microenvironment in vitro. CD3+ T cells were obtained by CD3+ cell selection system and the proportions of CD4+CD8- and CD4-CD8+ cells were measured by flow cytometry. The proliferation activity of T cells was measured by MTT colorimetry in both spontaneous proliferating group and superantigen (SAg)-stimulated group. And the levels of IL-4, IL-8 and IFN-? were quantified by enzyme-linked immunosorbent assay in culture supernatant. Results Mature CD4+CD8- cells and CD4-CD8+ cells were detected from CD3+ cells, which were derived from bone marrow CD34+ cells. When the psoriatic patients and normal controls were compared, there was no significant difference in the proportion of CD4+CD8- cells or that of CD4-CD8+ cells in CD3+ cells. The proliferation activity of T cells was significantly higher in the psoriatic patients than that in the normal controls. There was no statistical difference of IL-4?IL-8 or IFN-? levels in the supernatant of T cells between the spontaneous group and the normal control. However, the IFN-? and IL-8 levels in the supernatant of T cells were higher in the psoriatic patients than those in the normal controls after the SAg stimulation. Conclusion The abnormal activity of peripheral blood T cells from psoriatic patients with family history may be related to the bone marrow hematopoietic cells.

Objective:To investigate the colony formation of high-proliferative potential colony-forming cells(HPP-CFC) from bone marrow-derived hematopoietic cells of psoriatic patients and p21 gene promotor methylation in HPP-CFC,and probe into the relationship between the colony formation and the methylation status of p21 gene promoter.[WT5"HZ] Methods:[WT5"BZ]Bone marrow-derived mononuclear cells were separated by density gradient centrifugation.The cells were cultured in methycellulose semi-solid culture medium with SCF,GM-CSF,IL-3 and IL-6 for 14 days, and then high-proliferative potential colony-forming cells(HPP-CFC) were counted.The HPP-CFC were collected and their genomic DNA was isolated . DNA was subjected to bisulfite treatment,and the modified DNA was studied by using the methylation-specific polymerase chain reaction (MSP).[WT5"HZ]Results:[WT5"BZ]In methycellulose semi-solid culture system, the number of HPP-CFC in bone marrow of psoriatic patients was significantly less than that of normal control. The positive frequency of methylation of p21 gene promoter in HPP-CFC of normal contrasts was higher than that of psoriatic patients. [WT5"HZ]Conclusion:[WT5"BZ]The activity of methylation status of p21 gene promoter of bone marrow derived hematopoietic cells of psoriatic patients is abnomal. The lower positive frequency of methyllation of p21 gene promotor in HPP-CFC perhaps play a role in lower colony-forming capability of HPP-CFC of psoriatic patients.

Objective:To reveal the action of psoriatic peripheral blood T lymphocytes on keratinocytes proliferation and the significance in the pathogenesis of psoriasis.Methods:Keratinocytes were cocultivated with psoriatic peripheral blood T lymphocytes in comparison with those cocultivated with normal T lymphocytes. Immunohistochemical technique was performed to detect the expression of Ki67, c-Myc and Bcl-xL proteins.Results:There were significant overexpression of Ki67, c-Myc and Bcl-xL proteins in keratinocytes cocultivated with psoriatic T lymphocytes compared with the uncocultivated group and normal T lymphocytes group. Expression of Ki67, c-Myc and Bcl-xL proteins in keratinocytes cocultivated with normal T lymphocytes were not significantly different from the uncocultivated group.Conclusion:Psoriatic T lymphocytes, which have specific activity, can induce keratinocytes abnormal pattern of proliferation. One of the important mechanisms might be its action on the expression of c-Myc and Bcl-xL proteins.