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Background and Objectives@#Mesenchymal stem cells (MSCs) have immunomodulatory function and participate in the pathogenesis of many immunoregulation-related diseases, including psoriasis. Previously, we found that MSCs from psoriatic lesions overexpress the proinflammatory microRNA, miR-155 and exhibit a decreased immunosuppressive capacity. But the origin of these aberrant characteristics is still not clear. To investigate whether inflammatory cytokines in serum and peripheral blood mononuclear cells (PBMCs) from psoriatic patients can regulate the expression patterns of immunoregulation-related cytokines and the immunoregulation function of MSCs. @*Methods@#and Results: Normal dermal mesenchymal stem cells (nDMSCs) were treated with serum or PBMCs derived from patients with psoriasis or healthy donors. Expression of miR-155 and immunoregulation-related genes in each MSCs were measured using real-time PCR or western-blot. Meanwhile, the immunosuppressive capacity of DMSCs was evaluated by its inhibitory ability on proliferation of activated PBMCs. Compared to control serum, psoriatic serum significantly increased the expression levels of miR-155 (27.19±2.40 vs. 3.51±1.19, p<0.001), while decreased TAB2 expression (0.28±0.04 vs. 0.72±0.20, p<0.01) in DMSCs. Expression levels of immunoregulation-related genes such as PGE2, IL-10, and TLR4 were also markedly down-regulated following the psoriatic serum treatment. Those DMSCs treated with healthy serum could inhibit PBMC proliferation, while those psoriatic serum-treated DMSCs could not inhibit PBMC proliferation effectively. @*Conclusions@#Psoriatic serum up-regulate the expression of miR-155, down-regulate the expression of immunoregulation- related genes (PGE2, IL-10, and TLR4) in DMSCs, and along with the inhibition of the immunosuppressive function of MSCs.
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Background and Objectives@#Psoriasis is a chronic inflammatory skin disease, which the mechanisms behind its initiation and development are related to many factors. DMSCs (dermal mesenchymal stem cells) represent an important member of the skin microenvironment and play an important role in the surrounding environment and in neighbouring cells, but they are also affected by the microenvironment. We studied the glucose metabolism of DMSCs in psoriasis patients and a control group to reveal the relationship among glucose metabolism, cell proliferation activity，and VEC (vascular endothelial cell) differentiation in vitro, we demonstrated the biological activity and molecular mechanisms of DMSCs in psoriasis. @*Methods@#and Results: We found that the OCR of DMSCs in psoriatic lesions was higher than that in the control group, and mRNA of GLUT1 and HK2 were up-regulated compared with the control group. The proliferative activity of DMSCs in psoriasis was reduced at an early stage, and mRNA involved in proliferation, JUNB and FOS were expressed at lower levels than those in the control group. The number of blood vessels in psoriatic lesions was significantly higher than that in the control group (p＜0.05), which the mRNA of VEC differentiation, CXCL12, CXCR7, HEYL and RGS5 tended to be increased in psoriatic lesions compared to the control group, in addition to Notch3. @*Conclusions@#We speculated that DMSCs affected local psoriatic blood vessels through glucose metabolism, and the differentiation of VECs, which resulted in the pathophysiological process of psoriasis.
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Development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (S{Delta}C-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of S{Delta}C-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with S{Delta}C-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.
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Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome1, 2. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides3-6. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 [A], presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.
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Objective:To culture and identify dermal mesenchymal stem cells (DMSCs) in skin lesions of patients with psoriasis, and to determine the expression of hairy and enhancer of split-1 (HES1) and chemokine ligand 6 (CXCL6) in DMSCs.Methods:DMSCs were isolated from skin lesions of 15 patients with psoriasis and normal skin tissues of 18 healthy controls, and then subjected to culture. Cell phenotypes were identified by flow cytometry, and mRNA and protein expression of HES1 and CXCL6 was determined by real-time fluorescence-based quantitative PCR (RT-PCR) and Western blot analysis respectively. Comparisons were performed between 2 groups by using t test. Results:There was no difference in the morphology of DMSCs between the psoriasis group and control group. The mRNA expression of HES1 and CXCL6 in the psoriasis group was 3.56 and 3.44 times that in the control group respectively, and there was a significant difference between the two groups (both P < 0.05) . The protein expression of HES1 and CXCL6 in DMSCs was significantly higher in the psoriasis group than in the control group (both P < 0.05) . Conclusion:The high expression of HES1 and CXCL6 in DMSCs from lesions may be involved in the occurrence of psoriasis.
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Objective To investigate the characteristics of self perceived burden (SPB) in patients with chronic obstructive pulmonary disease (COPD) and explore the relationship between the perceived burden and self-efficacy,coping style.Methods A total of 96 cases with COPD in our hospital from January 2015 to June 2017 were enrolled in the study.The clinical data of patients were retrospectively analyzed.The patients were surveyed by self perceived burden scale,self-efficacy scale and coping style scale.The relationship between self perceived burden and self efficacy,coping style were investigated.Results The self perceived burden score of COPD patients was (33.76 ± 7.65) points with moderate level.The symptom management self-efficacy,common disease management self-efficacy score of COPD patients were (8.13 ± 1.09) and (8.22 ± 1.13) respectively with moderate self-efficacy.The total score of coping style in COPD patients was (35.09 ± 10.83) points with positive coping style.The each dimension and the total score of self perceived burden were negatively correlated with positive coping style in COPD patients (P ＜ 0.05),and positively correlated with negative coping styles (P ＜ 0.05).The total score of coping style had no significant correlation with perceived burden scores (P ＞ 0.05).The each dimension and the total score of self perceived burden were negatively correlated with the each dimension scores and total scores of self-efficacy in COPD patients (P ＜ 0.05).Conclusions There are significant correlation between SPB and self-efficacy and coping style in COPD patients.Medical staff should strengthen communication with patients,and guide them with positive coping style in the face of disease,which can effectively improve their self-efficacy and reduce the self perceived burden and promote physical and mental health.
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Objective To investigate the role of circular RNA (circRNA) in the occurrence of psoriasis.Methods Mesenchymal stem cells were isolated from skin lesions of 15 patients with psoriasis (psoriasis group) and skin tissues of 15 healthy human controls (control group) separately,and then subjected to cultivation.Flow cytometry and cell differentiation assay were performed to identify these mesenchymal stem cells.RNA sequencing was conducted to measure the expression of circRNA,and detailed bioinformatics analysis was performed.Then,7 differentially expressed circRNAs were selected,and a circRNA-microRNA interaction network was established.In this network,3 related microRNAs were selected and validated by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR).Statistical analysis was carried out by a two-sample t-test for comparison of qRT-PCR results between the psoriasis group and control group.Results As RNA sequencing showed,a total of 6 323 circRNAs were identified,and 3 227 out of these circRNAs were first reported in this study.Compared with the control group,129 circRNAs were differentially expressed in the psoriasis group,including 123 up-regulated circRNAs and 6 down-regulated circRNAs.The predicted circRNA-microRNA interaction network showed that several psoriasis-related microRNAs were associated with the 7 circRNAs,such as miR-17-5p,miR-30e-5p,miR-142-3p/5p,miR-369-3p,miR-184,miR-4490,miR-654-3p and miR-423-5p.qRT-PCR also confirmed that the 7 differentially expressed circRNAs were up-regulated in the psoriasis group.Compared with the control group,the expression of the 3 selected microRNAs significantly decreased in the psoriasis group (t =3.993,3.217,2.918,respectively,all P ＜ 0.05).Conclusion There is aberrant expression of circular RNAs in the mesenchymal stem cells from psoriatic skin lesions,which may take part in the occurrence of psoriasis.
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Objective To establish a method for isolating and culturing fibroblasts from cirrhotic liver in vitro,observe the biological characteristics of fibroblasts.Methods Human fibroblasts from cirrhotic liver were isolated and cultured by adhering to the culture plastic.The morphologic and growth characteristics of the acquired cells were observed by microscopy imaging and cell counting.The expression of FSP-1 and Vimentin of fibroblasts was detected through immunofluorescence and western blotting.Results The tissue blocks were well-adherent to the culture plastic within 2 hours.Hepatocyte-like cells were observed surrounding the blocks within about 1 week,and spindle-shaped fibroblasts were observed within about 2 weeks.After a series of passage,the attached cells became proliferated quickly.The growth curves of the passage 3,4 and 5 were quite similar.The result of immunofluorescence showed the expression of FSP1 and Vimentin was positive.The positive rate was respectively (90.6 ± 1.0) ％ and (91.2-± 4.1) ％.Conclusions Human fibroblasts from cirrhotic liver can be cultured successfully through adherent property.The method for isolation and culture of fibroblasts from cirrhotic liver is convenient,efficient,stable and cultured cells remain naive biological characteristics.
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Objective To evaluate the inhibitory effect of mesenchymal stem cells (MSCs) in lesions of patients with psoriasis on T lymphocyte proliferation.Methods Tissue specimens were obtained from the lesions of 15 patients with psoriasis vulgaris (7 at progressive stage and 8 at resting stage) and normal skin of 15 human controls from the Department of Urology and Plastic Surgery,Taiyuan City Centre Hospital.MSCs were isolated from these skin specimens,cultured,and identified using flow cytometry and in vitro differentiation assay.Enzyme-linked immunosorbent assay (ELISA) was performed to detect the concentration of interleukin (IL)-6,IL-1 1,hepatocyte growth factor (HGF) and transforming growth factor (TGF)-β1 in the culture supernatant of third-passage MSCs.Peripheral blood T cells were obtained from a healthy adult and cocultured with the third-passage MSCs for four days.Then,cells were counted and methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate the proliferation of T cells.One-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) test were carried out to compare the proliferation of T lymphocytes,and two independent samples t test to compare the concentrations of cytokines.Results Inverted microscopy revealed that the patient-and control-derived MSCs shared similar morphological properties and multi-directional differentiation capacity,along with the expression of CD29,CD44,CD73,CD90 and CD105,but absence of CD34,CD45 and HLA-DR on cell surface.After coculture with MSCs from the patients and controls for four days,the count of T lymphocytes per milliliter was (1.67 ± 0.34) × 105 and (1.04 ± 0.29) × 105 respectively (P＜ 0.01),and the proliferative activity (expressed as absorbence at 492 nm)was 0.317 ± 0.021 and 0.275 ± 0.007 respectively (P ＜ 0.01).Compared with the control-derived MSCs,the patient-derived MSCs showed a significantly higher level of IL-1 1 ((181.37 ± 31.74) vs.(130.07 ± 29.20) ng/L,t =5.32,P ＜ 0.01),but a lower level of lL-6 ((61.67±17.53) vs.(76.74±18.96) ng/L,t=2.61,P＜0.05)and HGF ((319.24 ± 41.03) vs.(352.35 ± 51.47) ng/L,t =2.25,P＜ 0.05),as well as a similar level of TFG-β1,in the culture supernatant.Conclusions The inhibitory effect of MSCs in psoriatic lesions on T lymphocyte proliferation is diminished,which may contribute to the pathogenesis of psoriasis.
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[Objective] To assess the changes in bone marrow microenvironment in patients with psoriasis by determining the level of transforming growth factor β1 (TGF-β1),stem cell factor (SCF),keratinocyte growth factor (KGF) and tumor necrosis factor-α (TNF-α) secreted by bone marrow stromal cells(BMSCs).[Methods] This study recruited 20 healthy controls with normal bone marrow picture and 20 patients with psoriasis vulgaris,including 10 at progressive stage and 10 at resting stage.The psoriasis area and severity index (PASI) score varied from 0.6 to 22.8 and averaged 10.97 in these patients.Bone marrow mononuclear cells were isolated by density-gradient centrifugation from bone marrow of these subjects,and BMSCs were cultured with adherent method.After three passages,the BMSCs were subjected to a 72-hour culture followed by the identification of cell phenotypes via flow cytometry and determination of TGF-β1,SCF,KGF and TNF-α levels in the culture supernatant via enzyme linked immunosorbent assay (ELISA).The parameters were compared by two independent samples t test between the two groups,and the correlation of eytokines with PASI was assessed by Pearson correlation analysis.[Results] Inverted microscopy revealed no obvious difference in the morphology of BMSCs between the patients and controls.CD29 was expressed by more than 90％ of the BMSCs,but no expression of CD45,CD34 or HLA-DR was observed in them.The BMSCs from patients showed a significantly lower level of supematant TNF-α ((22.93 ± 10.1 1 ) μg/L vs.(35.73 ± 15.15) μg/L,t =3.14,P ＜ 0.05),a higher level of supernatant SCF ((76.80 + 16.19) μg/L vs.(59.86 + 22.41) μg/L,t =2.74,P＜ 0.05),and asimilar level of supernatant KGF and TGF-β1 (both P＞ 0.05) compared with those from the controls.The PASI score was uncorrelated with the levels of SCF,TNF-α,KGF or TGF-β1 secreted by BMSCs in patients with psoriasis (all P＞ 0.05).[Conclusion]s The levels of SCF and TNF-α secreted by BMSCs are aberrant in patients with psoriasis,hinting an abnormal bone marrow microenvironment in these patients.
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Objective To study the expressions of Notch receptors (Notch1 and 2) and target gene Hes-1 in bone marrow CD34+ cells from patients with psoriasis. Methods CD34+ cells were isolated from the bone marrow of 12 patients with psoriasis and 19 normal human controls. Western blot was conducted to detect the expressions of Notchl, Notch2 and Hes-1 proteins in CD34+ cells, with the house-keeping gene β-actin as an internal reference. Results In contrast with normal controls, a significant increment was observed in the expressions of Notch1 and Hes-1 proteins in psoriatic patients (0.9488 ± 0.3221 vs. 0.6693 ± 0.1465, 0.8579 ±0.3729 vs. 0.5728 ± 0.1787, both P ＜ 0.05). No significant difference was observed in the expression of Notch2 between psoriatic patients and normal controls (0.7568 ± 0.3461 vs. 0.8312 ± 0.2887, P ＞ 0.05).Conclusion The overexpression of Notch1 and Hes-1 may be associated with the low proliferation activity of psoriatic hematopoietic cells.
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Objective To assess the preferential expressions of peripheral blood T cell receptor beta chain variable region (TRBV) subfamilies in patients with psoriasis vulgaris(PV), and to estimate their role in the pathogenesis of psoriasis. Methods Thirty-three upstream primers were designed to target the human functional TRBV genes, downstream primers to target the common T cell receptor beta constant (TRBC) gene,with T cell receptor alpha constant (TRAC) gene as the internal reference. Total RNA was extracted from the peripheral blood T cells of 10 health human controls and 10 patients with PV, and transcribed into cDNA.Then, TRBV genes were amplified by real-time fluorescence quantitative PCR (RFQ-PCR) and the fluorescence intensity of each samples was detected. The expression levels of TRBV genes in the control group were used to calculate the cut-off values (mean expression levels of TRBV subfamilies in the 10 normal controls + 3 standard deviations). When the expression level of a TRBV subfamily from patients with PV was equal to or higher than the cut-off value, it was considered as the preferentially expressed TRBV subfamily. Results The threshold cycle (Ct) value varied from 21 to 24 for TRAC gene. The difference in the Ct value between TRBV subfamily genes and TRAC gene in patients with PV was 2.98 for TRBV2 gene, 3.24 for TRBV5-7 gene, 2.52 for TRBV6-6/6-9 gene, 2.04 for TRBV 12 gene, 3.56 for TRBV 24 gene, and 4.12 for TRBV 29 gene, and the expression levels of these subfamily genes were significantly higher than those in the normal controls (all P ＜ 0.05). According to the above standard, TRBV6-6/6-9, TRBV12 and TRBV29 were considered to be preferentially expressed subfamilies. Conclusions There is a preferential expression of TRBV gene subfamilies in peripheral blood of patients with psoriasis vulgaris, which may play a vital role in the abnormal T cell-mediated immune responses in psoriasis.
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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.
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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.
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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
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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.
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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
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Objective:To reveal the relation between RUNX1 and hematopoietic cells by examination of the expression of RUNX1 and its target gene SLC9A3R1 in bone marrow CD34+ cells from patients with psoriasis,as well as the RUNX1 linkage locus between SLC9A3R1 and NAT9.Methods:Bone marrow CD34+ cells were isolated from psoriatic patients and normal persons by immunomagnetic cell selection.Expression of mRNA for RUNX1 and SLC9A3R1 were analyzed using reverse transcriptase-polymerase chain reaction(RT-PCR),the RUNX1 linkage locus between SLC9A3R1 and NAT9 was detected.Results:The positive frequency of RUNX1 in bone marrow CD34+ cells from patients with psoriasis was lower than in normal controls(P
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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.
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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.
