0.05). CONCLUSION: Both modified Dexter method and IMDM medium method can promote the adherence and proliferation of bone marrow stromal cells in a short time. It is the simple and practical culture method of bone marrow stromal cells by culturing the cells in the medium of IMDM compared to the modified Dexter method.

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 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 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 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 investigate the mechanism of abnormal epidermal proliferation induced by psoriatic T lymphocytes.Methods Skin organ culture was established with psoriatic T cells mixed with epi-dermal cells.The expressions of c-myc,bcl-2and p53protein were examined with immunohistochemical method in epidermis before culture,on the3rd day and the6th day after culture.Results Significant up-regulation of c-myc and p53protein was found in psoriatic lesions,but bcl-2protein expression was rarely observed.The expressions of those proteins were normal in non-lesional psoriatic skin.The p53protein ex-pression was increased in normal skin and non-lesional psoriatic skin on the3rd day after culture with psori-atic T cells,and c-myc protein expression was enhanced while bcl-2was decreased on the6th day of co-culture.There was no significant difference of those proteins' expression between normal skin and non-lesion-al psoriatic skin stimulated by psoriatic T cells.Conclusions The abnormal expressions of c-myc,bcl-2and p53protein play an important role in abnormal epidermal proliferation and differentiation in psoriasis.Psoriatic T lymphocytes can influence c-myc,bcl-2and p53protein expression in normal skin and non-le-sional psoriatic skin.Pathogenic T cells rather than keratinocytes might be vital for initiation of psoriasis.

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 of isolating culture and differentiation of human bone marrow-derived mesenchymal stem eeUs into blood vessel endothelial-like cells in a specialized micro-environment in vitro,SO as to provide an experimental foundation for psoriasis.Methods:The hMSCs were isolated by density gradient centrifugation,amplificated and identificated in vitro.Vascular endothelial growth factor (VEGF)and basic fibroblast growth factor(bFGF)within endothelial cell growth medium(DMEM)were used to induce hMSCs differentiation into vascular endothelial-like cells.The induced hMSCs were detected by flow cytometry to find whether they had endothelial cell phenotypes.The Dil-ac-LDL ingestion assay Was used to apprmses the blood vessel endothelial-like cell function.Results:In cell morphology,the induced hMSCs transformed into endothelial-like cells.These cells expressed specific surface markers of,Vascular endothelial-like cells such as CD34,CDl06,HLA-DR,CD54,VWF,CD31,KDR and CD5 comparing to those in the control group(P＜0.01).The induced endothelial-like eeHs had the ability of ingesting Dil-ac-LDL.Conclusion:Combination of Density gradient eentrifugation and adherent methods can obtain pure MSCs.hMSCs Can obtain endothelial cell phenotypes after induced by VEGF and bFGF in vitro.Human hnSCs have potential to differemiate into vascular endothelial-like cells.The induced endothelial-like cells have completely mature endothelial cell functional properties.

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 investigate the effects of psoriatic keratinocytes on the expression of CD25 and CD69 in T lymphocytes. Methods Keratinocytes were isolated from the biopsy samples resected from the lesions and adjacent non-lesional area of 10 patients with psoriasis, and cultured in 5% CO2 at 37 ℃ in 24-well plates. Density gradient centrifugalization and glass adherence method were applied to detach peripheral blood mononuclear cells (PBMC) and peripheral blood T lymphocytes (PBTL) from anticoagulant blood samples of the same 10 psoriatic patients and 10 normal controls. PBMCs of 1×105/well were added to the wells containing cultured keratinocytcs of 1×105/well, then gamma rays were used to inactivate these cells. Following that, PBTLs of 1×106/well were inoculated into the 24-well plate containing inactivated keratinocytes and PBMCs, and cultured in 5% CO2 at 37 ℃. Those PBTLs cultured without the presence of keratinocytes or PBMCs served as the natural growth control. Three days later, flow cytometry was performed to detect the expression of CD25 and CD69 in PBTLs. Results There was a significant increase in the expression of CD25 and CD69 in psoriatic PBTLs cocultured with lesional kcratinocytes compared with those cocultured with non-lesional keratinocytes and natural psoriatic controls. Also, the expression of CD25 and CD69 was increased in normal PBTLs cocultured with lesional or non-lesional keratinocytes of psoriatc patients than those in the natural normal controls. No significant differenco was observed in the expression of CD25 or CD69 between psoriatic PBTLs cocultured with non-lesional keratinocytes and natural psoriatic PBTLs, or between the normal PBTLs cocultrred with lesional keratinocytes and those with non-lesienal keratinocytes (P>0.05). Conclusions Psoriatic keratinocytes may act as an autoantigen to trigger autoimmune response and eventually lead to a chronic local inflammation in patients with psoriasis.