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1.
AIM: To investigate the role of prostaglandin E2 receptor 2 agonist (EP2A) in proliferation and homing of human CD34+ cells. METHODS: Bone marrow fluid and peripheral blood containing stem cells were collected from healthy donors mobilized by granulocyte colony-stimulating factor in our department. Human CD34+ cells were isolated by the method of magnetic-activated cell sorting microbeads. Bone marrow mononuclear cells were isolated by Ficoll-Paque centrifugation, and the bone marrow mesenchymal stem cells (BMMSC) were cultured with L-DMEM. Human CD34+ cells and BMMSC were divided into 4 groups, and treated with PGE2 (as positive control), DMSO (as negative control), EP2A and EP2A+prostaglandin E2 receptor 2 antagonist (EP2AA), respectively. After exposed to the reagents, human CD34+ cell viability was measured by CCK-8 assay, the number of colonies was evaluated by colony-formation assay, the cell cycle distribution was analyzed by flow cytometry, and the protein expression of survivin, β-catenin and CXC chemokine receptor 4 (CXCR4) was detrmined by Western blot. Moreover, the concentration of stromal cell-derived factor-1α (SDF-1α) in the BMMSC was detected by ELISA. RESULTS: The cell viability and the colony number of human CD34+ cells in EP2A group were not higher than those in negative control group. Furthermore, the proportion of human CD34+ cells treated with EP2A in G2/M phase was not elevated compared with negative control group. The protein expression of survivin and β-catenin did not up-regulated in human CD34+ cells exposed to EP2A, but the protein expression of CXCR4 in human CD34+ cells and the concentration of SDF-1α in BMMSC were elevated. CONCLUSION: EP2A promotes human CD34+ cell homing in vitro but not proliferation.  相似文献   

2.
AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4+CD25+ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4+CD25+ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4+CD25+T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4+CD25+ T cells in CD4+T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4+CD25+ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4+CD25+ regulatory T cells.  相似文献   

3.
AIM: To explore the effects of romidepsin (FK228), a novel histone deacetylase inhibitor, on the effector and regulatory T cells in vitro.METHODS: As the reactive cells, lymphocytes, CD4+ T cells and CD8+ T cells were labelled with CFSE, and stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group), or PBS (placebo group).After 72 h, the proliferation of the cells was detected in different groups. The lymphocytes were stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group),or PBS (placebo group). After 72 h, the percentage of CD4+ Foxp3+ T cells and the levels of related cytokines were detected in different groups. RESULTS: The proliferation of CFSE-labelled lymphocytes, CD4+ T cells and CD8+ T cells triggered by anti-CD3 and anti-CD28 mAbs all were inhibited when cultured with romidepsin at concentrations of 1 μmol/L, 3 μmol/L and 5 μmol/L in a dose-dependent manner (P<0.05). Compared with placebo group, in the presence of anti-CD3 and anti-CD28 mAbs, 1 μmol/L romidepsin did not increase the percentage of CD4+ Foxp3+ T cells (P>0.05). When cultured with romidepsin at concentrations of 3 μmol/L and 5 μmol/L, the percentage of CD4+ Foxp3+ T cells was enhanced markedly (P<0.05). The levels of IL-10 and TNF-α in the supernatant were markedly increased in positive control group and 3 experimental groups (P<0.05), and the levels of cytokines in different experimental groups were gradually decreased with the elevation of FK228 concentration (P<0.05). The level of TGF-β was slightly increased in positive control group with no significant difference compared with placebo group (P>0.05). With the increase in the concentration of FK228 in different experimental groups, the TGF-β level was increased in a dose-dependent manner and there were significant differences in the 3 experimental groups. Meanwhile, significant differences existed between experimental groups and placebo group and between experimental groups and positive control group (P<0.05). CONCLUSION: Romidepsin inhibits the proliferation of CD4+ and CD8+ effector T cells and increases the percentage of CD4+ Foxp3+ regulatory T cells. It may be related to the increased level of TGF-β, but independent of IL-10.  相似文献   

4.
AIM:To establish the method for detecting the immunophenotype of immunosuppressive receptor programmed cell death protein 1 (PD-1) in T-cell receptor (TCR) Vβ repertoire of CD3+, CD4+ and CD8+ T-cell subsets, therefore to evaluate the distribution of PD-1 in T-cell repertoire from human peripheral blood (PB). METHODS:The PB samples from 10 cases of healthy individuals (HI) were collected. Using multi-colored fluorescence flow cytometry, the distribution frequency of PD-1 in TCR Vβ repertoire was detected with a wide panel of anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-PD-1 and 24 anti-TCR Vβ repertoire (IOTest® Beta Mark TCR Vβ Repertoire Kit, containing 8 tubes which labeled A~H, each tube is a composite antibody of FITC and PE coupling, each cocktail contains antibodies direc-ted to 3 different Vβ subfamilies) monoclonal antibodies. RESULTS:The total number of the 24 TCR Vβ repertoire detected in CD3+, CD3+CD4+ and CD3+CD8+ T cells from 10 cases of HI was consistent with the Quick Reference Card data provided by the kit. The preliminary results showed that the frequency of Vβ usage in CD3+, CD4+ and CD8+ T cells was different. High usage of Vβ2, Vβ3, Vβ8, Vβ9, Vβ5.1, Vβ13.1 and Vβ13.2 was found in CD3+ T cells, while high usage of Vβ2, Vβ3, Vβ8, Vβ5.1, Vβ9 and Vβ13.1 in CD3+CD4+ T cells, and high usage of Vβ1, Vβ2, Vβ3, Vβ9, Vβ13.1 and Vβ13.2 in CD3+CD8+ T cells were also observed. Further analysis showed that the expression of PD-1 was detected in all 24 TCR Vβ subfamilies of CD3+, CD3+CD4+ and CD3+CD8+ T cells. The higher frequency of PD-1+ T cells was CD4+Vβ5.2+ T cells, whereas the higher frequency of PD1+ T cells in CD8+Vβ11+ and CD8+Vβ13.6+ T cells was detected. CONCLUSION:The method for detection of the immunosuppressive receptor PD-1 in TCR Vβ repertoire of T-cell subsets is successfully established, which provides a new method for further analysis of immunosuppressive characteristics of TCR Vβ repertoire in the patients with leukemia.  相似文献   

5.
AIM: To explore the correlation between development of CD4+CD25+ regulatory T cells (CD4+CD25+ Tr) and thymus CD4-CD25+ cells. METHODS: The ratios of CD4+CD25+ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4-CD25+ cells to CD4- T cells in thymus were measured by flow cytometry. Purified CD4+CD25+ T cells and CD4+CD25- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4+CD25+ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4-CD25+ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4+CD25+ Tr and CD4+CD25- T cells showed no proliferation in response to ConA, while CD4+CD25+ Tr showed a transient enlargement of cell size. Both CD4+CD25+ Tr and CD4+CD25- T cells underwent proliferation in response to PDB plus ionomycin. CD4+CD25- T cells, but not CD4+CD25+ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4+CD25+ Tr showed significant proliferation and CD4+CD25- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4-CD25+ cells are probably the precursor of CD4+CD25+ Tr during cell development.  相似文献   

6.
AIM: To investigate the role of Th17 cells in the patients with cervical cancer.METHODS: We measured the peripheral levels of Th17 cells and CD3+CD8-IL-21+ T cells in 37 cervical cancer (CC) patients, 25 cervical intraepithelial neoplasia (CIN) patients and 18 healthy controls by flow cytometry. The percentages of Th17 cells and CD3+CD8-IL-21+ T cells in total CD4+ cells were calculated.RESULTS: Compared with controls, the patients with CC or CIN had higher proportions of Th17 cells (all P<0.01) and CD3+CD8-IL-21+ T cells (all P<0.05). Notably, in CC patients, the increased percentages of Th17 cells and CD3+CD8-IL-21+ T cells were independently associated with the clinical stage(all P<0.05), lymph node metastasis (P<0.01,P<0.05) and vasoinvasion (all P<0.01), while the elevated percentage of CD3+CD8-IL-21+ T cells was also associated with the tumor size(P<0.01). Furthermore, the percentage of Th17 cells was positively correlated with that of CD3+CD8-IL-21+ T cells in healthy controls and CC patients, but not in CIN patients.CONCLUSION: Our results indicates a possible role of Th17 cells in CC patients correlated with CD3+CD8-IL-21+T cells, and the elevated percentage of circulating Th17 cells may be involved in the development and progression of cervical cancer.  相似文献   

7.
AIM: To investigate the effect of Thymosin α1 on the development and matutation of thymocytes. METHODS: The proportion of CD4+CD8+ thymocytes and the expression of smoothened (Smo) of the hedgehog (Hh)-signaling in CD4-CD8-thymocytes were examined to observe the effect of thymosin α1 on thymocyte development and matutation. RESULTS: Flowcytometric analysis showed that thymosin α1 showed activity at a low dose of 30 μg/kg, and 30 μg/kg thymosin α1 accelerated the replenishment and maturation of thymocytes according to the expression of Smo of the Hh-signaling in CD4-CD8-thymocytes, the potent negative regulator of proliferative responses. CONCLUSION: Thymosin α1 can accelerates thymocyte development from CD4-CD8- to CD4+CD8+.  相似文献   

8.
AIM: To investigate how human adipose-derived stem cells (hASCs) regulates the differentiation of Th17 cells in multiple sclerosis. METHODS: hASCs were isolated from the adipose tissues. Magnetic-activated cell sorting (MACS) kit was used to isolate CD4+ T cells from peripheral blood mononuclear cells (PBMCs) which were isolated by density gradient centrifugation. The percentage of CD4+ T cells was detected by flow cytometry. The activated CD4+ T cells were co-cultured with hASCs for about 4 d at different ratios of hASCs to CD4+ T cells (1:4 and 1:10) in a Th17 polarised condition. Another group adding anti-leukemia inhibitory factor (LIF) antibody was set up. Th17 cell proportion of the CD4+ T cells was determined by flow cytometry. The level of LIF in the supernatant of co-cultured system was measured by ELISA. The mRNA expression of retinoid-related orphan receptor γt (RORγt), interleukin-6 receptor (IL-6R), interleukin-23 receptor (IL-23R), LIF and leukemia inhibitory factor receptor (LIFR) was detected by real-time PCR. RESULTS: The result of flow cytometry suggested there were mainly hASCs, and the percentage of CD4+ T cells in the PBMCs were above 90% after MACS. The Th17 cell proportion decreased in 1:4 and 1:10 co-cultured groups in a dose-dependent manner. The mRNA expression of IL-6R, IL-23R and RORγt was downregulated and the expression of LIFR and LIF was up-regulated. When the anti-LIF was added into the co-cultured system, the ratio of Th17 cells increased and reached to the control level. The protein level of LIF obviously increased after co-cultured. After anti-LIF added, the mRNA expression of RORγt and IL-6R was up-regulated. CONCLUSION: hASCs inhibits the differentiation of Th17 cells from multiple sclerosis patients through the competitive inhibition of LIF/IL-6 by secreting LIF.  相似文献   

9.
AIM: To characterize the proportion of CD14+CD16+ monocytes in peripheral blood from type 2 diabetes (T2DM) patients and to observe the response of CD14+CD16+ monocytes to lipopolysaccharide (LPS) and interleukin-15 (IL-15) for further exploring the potential mechanism of inflammatory immune response in the pathogenesis of T2DM. METHODS: Twenty-eight patients with T2DM and 20 healthy volunteers were enrolled in the study. The peripheral blood was collected for determining the percentage of CD14+CD16+ monocytes by flow cytometry. The peripheral blood mononuclear cells (PBMC) were isolated and subject to stimulation with LPS and IL-15 for 4 h. The protein expression of STAT5 was detected by Western blotting and the phosphorylated (p)-STAT5 was determined by Western blotting and immunofluorescence. Serum levels of 25-hydroxyvitamin D3 and IL-6, and the concentrations of IL-6 and monocyte chemoattractant protein-1(MCP-1) in the culture supernatants were assessed by ELISA. Serum level of C-reactive protein (CRP) was measured by immunoturbidimetry. RESULTS: There were positive correlations between the quantity of CD14+CD16+ monocytes and serum levels of CRP and IL-6 (r=0.394, P<0.05 and r=0.741, P<0.01), while serum 25 (OH) D3 was negatively correlated with the quantity of CD14+CD16+ monocytes (r=-0.409, P<0.01), serum CRP(r=-0.479,P<0.01) and serum IL-6 (r=-0.774,P <0.01). After stimulated with LPS and IL-15, PBMC showed significant up-regulation of p-STAT5 protein expression, and significant increases in the supernatant levels of IL-6 and MCP-1 were observed (P<0.05). The expression of p-STAT5 existed in the nucleus.CONCLUSION: These findings suggest that the functional disturbance in monocytes occurs in T2DM, which may be related to insufficiency of vitamin D3. The aberrant activation of STAT5 signaling pathway underlies the functional abnormalities of the monocytes in T2DM.  相似文献   

10.
AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4+ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4+T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4+T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4+T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4+ CD8- cells in the spleen and decreased the accumulation of CD4+ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4+ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4+ T cells of asthmatic mice.  相似文献   

11.
AIM: To Compare immunogenicity of three kinds of heterogenic corneal stroma. METHODS: 36 SD rats were randomized into 4 groups, each group consisting of 9 rats. Group 1 was control group. Three kinds of heterogenic corneal stroma: porcine, rabbit and chicken corneal stroma were heterotopically transplanted to subcutaneous layer of 27 (group 2-4) SD rats, respectively. The expression of CD4+, CD8+, CD25+, CD71+ on peripheral T cells was identified and analyzed by dual fluorescence flow cytometry at 7, 14, 28 days after operation. RESULTS: Compared with control group, the expression of CD4+, CD8+, CD25+, CD71+ was no significant change in porcine corneal stroma group(P＞0.05), the expression of CD4+ was increased in rabbit corneal stroma (P＜0.05), CD4+, CD4+ CD71+ markedly higher in the chicken corneal stroma (P＜0.01) at 7 days after operation. CONCLUSION: The immunogenicity of porcine stroma is the lowest in three kinds of heterogenic corneal stroma (chicken, rabbit and porcine).  相似文献   

12.
AIM:To study the role of cell membrane ectopic calreticulin (CALR) expression on the protective immunie effect of T-cell vaccine (TCV) on experimental autoimmune encephalomyelitis (EAE). METHODS:EAE model was established by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) immunization in C57BL/6 mice, and the mice were immunized with MOG35-55-specific CALR+ and CALR- T-lymphocytes. Symptomatic scores were compared at the maximum of the disease. On the 15th day after immunization, the proportion of CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in the spleen, and the expression of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10 and IL-17A in the serum were measured. RESULTS:Increased expression of CALR in activated T cells after γ-irradiation was observed. Blockade of CALR on the vaccinating T-cell surface reduced the protective effect of TCV. Furthermore, blockade of CALR reduced the number of Treg in the spleen and up-regulated pro-inflammatory cytokines. CONCLUSION:CALR expression in the T cells is necessary for the protective immunity induced by TCV in EAE mice.  相似文献   

13.
AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34+ and CD34- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34+ and CD34-,total MNC led to a significant increase in the expansion of CD34+ cells compared with CD34 enrichment (P＜0.05). There was a trend toward decreased apoptosis in cultures when early passage was performed once the linear cord like structures appeared. There was no significant effect on apoptosis between with VEGF and without VEGF group (P＞0.05). These differentiated EPCs were stained positive for CD34+, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34+ and AC133+cells accounted for 68.2%±6.3% (n=6) and 57.2%±9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34+ and CD34- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.  相似文献   

14.
AIM: To explore the relationship of circulating tumor cells (CTCs) with CD4+/CD8+, neutrophil-to-lymphocyte ratio (NLR), total tumor volume (TTV) and tumor stage in the patients with primary hepatocellular carcinoma.METHODS: We selected 80 cases of histologically diagnosed primary hepatocellular carcinoma in the study. The method of CanPatrolTM was used, which was developed by SurExam biotechnology company to identify CTCs in the blood. CD4+ T cells and CD8+ T cells were counted by flow cytometry. The patients were divided into 2 groups according to the levels of CD4+/CD8+ ratio, NLR and TTV. The patients were also divided into Ⅰ＋ Ⅱstage group and Ⅲ＋ Ⅳ stage group according to the seventh edition of TMN staging criteria.RESULTS: The numbers of peripheral blood CTCs and mesenchymal CTCs in high CD4+/CD8+ ratio group were significantly lower than those in low CD4+/CD8+ ratio group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs and hybrid CTCs in high TTV group were significantly higher than those in low TTV group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs and hybrid CTCs in I＋ II stage group were significantly lower than those in III＋ IV stage group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs, hybrid CTCs and epithelial CTCs between high NLR group and low NLR group had no statistical difference.CONCLUSION: CTCs exist in the peripheral blood of the patients with primary hepatocellular carcinoma. The peripheral blood CTCs have significant correlations with TTV, tumor stage and T-cell immunity.The worse cell immune function, the larger TTV and the later tumor stage a patient has, the more the peripheral blood CTCs are.  相似文献   

15.
AIM: Gene transduction with a recombined murine stem cell retroviral vector has been investigated to find an effective way of gene transduction and to offer theory and experimental basis for the recombined murine stem cell retroviral vector used for gene transduction. METHODS: 1. Construction of retrovirus vector: EC1-4 gene (repeats 1-4 of cadher in-5 extracellular domain) and mutant (Ser 222A) MEK1 gene were cloned into retrovirus vector pMSCV after cut by Bgl Ⅱ and EcoR 1 restriction endonuclease. 2. Obtaining CD41+ cells and cell culture: Cells expressing CD34+ from cord blood were isolated. The inducement of cells expressing CD41 from CD34+ cells was performed by using TPO and cells CD41+ were selected by FACS. NIH 3T3 cells were cultured in high sugar DMEM medium and U937 in RPMI 1640 medium. UT7 cells which is cytokine-dependent cell line were grown in Iscove's modified Dulbeco's medium supplemented by GM-CSF. 3. Determination of viral titers: Retroviral vectors were transferred to packaging cell line 293. Retroviral containing supenatant was collected after transfection. The viral titers was tested on infection of NIH 3T3 cells by FACS analysis. 4. Western blot: Transduced CD41+, UT7, U937 and MDA-MB-435 cells were analyzed by western blot to examine expression of transduced genes. RESULTS: A packaging cell line 293 produces high-titer MEK1 pMSCV retroviruses (3.1×107) and EC1-4 pMSCV retroviruses (1.0×108). With 8-folds dilution retroviruses, 60.73% GFP positive cells have been obtained in MEK1 pMSCV transduced UT7 cells, 72.56% in U937 cells and 30.57% in CD41+ cells, respectively. GFP positive cells have reached up 97.54 % in EC1-4 pMSCV transducted MDA-MB-435 cells. Phosphorylated MEK1 has been decreased in experiment group when TPO has stimulated CD41+ and UT7 cells or serum has stimulated U973 cells. This indicates that is a dominant negative effect of mutation MEK gene. EC1-4 gene transduced MDA-MB-435 cells have expressed EC1-4. CONCLUSION:The recombined murine stem cell retrovirus can effectively mediate gene transduction of CD41+,UT7,U937 and MDA-MB-435 cells,and transduced genes can be stably expressed.  相似文献   

16.
AIM:To investigate the biological characteristics of cytokine-induced killer (CIK) cellsin vitro.METHODS:The non-adhere peripheral blood monoclear cells from healthy donors were induced into CIK cells in the presence of IFN-γ, IL-1β, IL-2 and anti-CD3 antibody. LAK (lymphokine activated killer) cells were prepared as a control. The cellular phenotype were detected by FCM and immunocytochemistry and the cytotoxicity was measured by LDH release assay.RESULTS:After 2 weeks of induction, the proliferation rate of CIK cells reached a peak and the proportion of CD3+ population was above 95%, and then the cells growth entered to plateau phase at week 3. The proportion of CD3+CD56+ NKT subset cells was 16.5% on day 15 and it had no obvious variety between 2 and 4 weeks. Correspondingly, LAK cells grew slowly and had lower proliferation rate compared with the CIK cells (P<0.01). CIK cells showed higher specific lysis rates to BeL-7402 hepatoma cells than those of LAK cells at different effector to target ratio (P<0.01). Immunocytochemical staining showed the CIK cells highly co-expressed HLA-DR and CD54 antigens. The NKT cells were slightly bigger than CD3+CD56- cells and a large quantity of pseudopodia were observed on their surface.CONCLUSION:The CIK cells have higher proliferation potency and stronger cytotoxicity to lyse tumor cells than LAK cellsin vitro.Within the span of time from 14 to 21 days, the proliferation rate and the proportion of CD3+CD56+ subset of CIK cells all reach peaks. Therefore, CIK cells in this period are suitable for clinical application.  相似文献   

17.
LI Qian  SHEN Hua-hao 《园艺学报》2012,28(3):512-517
AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4+CD25+ Treg cells/CD4+ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4+CD25+Treg cells/CD4+ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4+CD25+ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.  相似文献   

18.
AIM:To investigate the details of age-related changes of T lymphocytes in order to seek for sensitive biomarker for immunosenesence.METHODS:Heparin anticoagulated venous blood was collected freshly from young (20-35 years) and elderly (50-75 years) volunteers and three or four color immunofluorescence staining was performed. The nucleated cells were acquired and the phenotypes of T lymphocyte subpopulations were analyzed with flow cytometry.RESULTS:There was no significant difference in percentages of pan-T (CD3+), helper T (CD4+) and cytotoxic (CD8＋) T subsets between young and elderly, whereas the density of CD3 molecule (MFI) on T cells in elderly group decreased significantly. It was also found that the rates of CD44+ and CD62L+ T cell subsets in young group did not have statistical difference from elderly. However, the rates of CD95+ pan-T, helper T and cytotoxic T subsets of elderly group were all markedly higher than that in young group.CONCLUSIONS:The relative rates of T cell and its subsets displayed no age-related changes while the density of CD3 was down-regulated during aging in these groups investigated. Moreover, the expression percentage of CD95 (Fas) on T cells increased as aging, suggesting that it is a potential biomarker for evaluating immunosenescence.  相似文献   

19.
AIM：To investigate new methods for the expansion of human CD8+ memory T cells in vitro and provide novel means of anti-viral and anti-tumor adoptive immunotherapy. METHODS：Six kinds of stimuli, anti-CD3 antibody, anti-CD28 antibody, CD70, IL-2, IL-7 and IL-15, for the expansion of human CD8+ memory T cells in vitro were selected and arranged for their combinations, resulting in 63 kinds of stimulating combinations. Normal human CD8+ T cells were isolated and exposed to these stimuli. After 14 days of cell culture, the number and purity of CD8+ T cells, and the percentages of CD8+ central memory T cells (TCM) and CD8+ effector memory T cells (TEM) were detected. The expansion folds of CD8+ T cells, CD8+ TCM and CD8+ TEM were calculated and the relatively better stimulating combination was determined. RESULTS：The combination of anti-CD3 antibodies, IL-2 and IL-7 was a better method for the expansion of human CD8+ T cells, CD8+ TCM and CD8+ TEM in vitro, and the expansion folds were 13.19, 13.28 and 15.27, respectively. CONCLUSION：The combination of anti-CD3 antibodies, IL-2 and IL-7 is the relatively better method for the expansion of human CD8+ memory T cells in vitro.  相似文献   

20.
AIM: To investigate the amount and patterns of expressing CD69, IL-4 and IFN-γ on TCRVα24+ NKT cells, and compare with that of CD3+ T cells from human peripheral blood in response to in vitro stimulation. METHODS: The whole blood was stained with three-color immunofluorescence directly or after cultured with PDB+ionomycin (Ion) for 6 h, then the mononuclear cells were separated by lysing red blood cells. The expression rates of CD69, IL-4 and IFN-γ on TCRVα24+ NKT cells and CD3+ T cells were estimated by flow cytometer. RESULTS: As a proportion of mature T cells, the ratio of TCRVα24+ NKT cells to CD3+ T cells was about (1.34±0.42)%. The expression rates of CD69 on TCRVα24+ NKT cells and CD3+T cells in response to PDB+Ion for 6 h were (96.71±1.33)% and (98.60±0.47)%, respectively, while the ratio were (11.47±2.86)% and (1.07±0.45)% in the unstimulated group, and there were significant difference between them. The expression rates of IL-4 and IFN-γ on TCRVα24+ NKT cells stimulated with PDB+Ion for 6 h were (48.62±2.44)% and (46.65±8.91)%, respectively, which were significantly higher than that of unstimulated group [(31.57±3.31)%, (13.45±6.29)%] and that of stimulated CD3+ T cells, though the expression rates on stimulated CD3+ T cells were significantly higher than that of unstimulated CD3+ T cells. CONCLUSION: There is small amount of NKT cells in adult human peripheral blood. The expression rates of IFN-γ and IL-4 on these lymphocytes are higher than CD3+ T cells, suggesting that NKT cells are important immunomodulatory cells in special microvironments.  相似文献   

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