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Objective To investigate the T cell cytotoxicity induced by recombinant adenovirus carrying HIV-1 vpr gene.Methods C8166 cells infected with rAd-vpr or negative control rAd-vector,were analyzed for cell cycle distribution and cell death by flow cytometry.The discrimination of living cells,apoptotic and necrotic cells were differentiated with Hoechst-PI double staining under the confocal microscopy.Changes of mitochondrial membrane potential(△ψm)were monitored by JC-1 staining method.Results Annexin V-PI and Hoechst-PI staining indicated the death effects of HIV-1 Vpr on C8166 cells.PI flow cytometric analysis showed that cell cycle arrested in G2 phase.C8166 cell△ψm collapse mediated by Vpr was detected by JC-1 fluorescent staining.Conclusion The ability of recombinant adenovirus carrying HIV-1 vpr gene to induce mitochondria dysfunction,cell cycle G2 phase arrest and cell death was confirmed in C8166 cells.
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Objective:To study the effect of isoflavone and genistein on activation of T lymphocytes in order to develope new immuno intervention reagent.Methods:Fluorescence conjugated monoclonal antibodies and flow cytometer were used to detect the expression rate of CD69 by activated T cells in vitro in response to Phytohemagglutinin(PHA) and Phorbol 12,13 dibutyrate(PDB),with some samples pre incubated with 10,50 or 100 ?mol/L of genistein,after 2 h and 6 h of incubation in whole blood culture system.Results:After 2 h of culture,the inhibitory effect in PHA group was stronger than PDB group(P
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AIM:To study the roles of bone marrow-derived dendritic cells from donor mouse treated with 17?-estradiol(E2)in immune tolerance induction in skin allograft.METHODS:Bone marrow-derived dendritic cells from C57 mouse as donor were cultured respectively treated with E2(E2 group).BALB/c mouse as recipient received respectively one injection of dendritic cells of E2 group,mature dendritic cell group and immature dendritic cell group intravenously.Skin transplantation was performed in the absence of immunosupression after 7 d.Mice that received PBS were served as control.The time of skin survival was observed after transplantation.Flow cytometry was used to analyze the percentage of CD4+CD25+ T cells in peripheral blood respectively before and after transplantation.RESULTS:Compared with immature dendritic cells and control group,the time of skin survival in E2 group was significantly longer(P
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AIM:To study the changes of mitochondria during apoptosis in Jurkat cells induced by arsenic oxide(As2O3).METHODS:By treated with 4?10-6 mol/L As2O3,apoptosis and necrosis of Jurkat cells were assessed by annexin V-FITC/PI double staining flowcytometry.Mitochondrial mass and its membrane potential(△?m)was measured by NAO/PI and DiOC6(3)/PI staining,respectively.Free radical formation was detected by DCFDA staining.RESULTS:After 48 h of As2O3 treatment,the rates of early apoptotic Jurkat cells in As2O3 and control groups were(18.98?1.40)% and(5.17?0.80)%,respectively(P
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AIM: To investigate the relationship between CD200~+CK7~+ trophoblasts and the resorption of embryos in a poly (I∶C)-induced abortion model. METHODS: The status of CD200 expression was investigated in Balb/c?C57BL/6 and Balb/c?Balb/c mice as induced model of embryo-resorption by an i.p. injection of poly (I∶C). CD200 expression on CK7~+ cells from placentas was detected with flow cytometry. CD200~+ cells in placenta were observed with immunocytochemical staining. RESULTS: Both the percentage and absolute number of CD200~+CK7~+ cells were dramatically decreased by injection of poly (I∶C) in Balb/c?C57BL/6 (6.3%?6.2% vs 36.1%?9.3%, P
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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.
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AIM: To study mitochondrial mass and structural protein changes in dexamethasone (DEX)-mediated mouse thymocyte apoptosis process. METHODS: DEX-induced mouse thymocyte apoptosis model was established. Annexin V-FITC/PI double staining was used to identify apoptotic and necrotic cells by flowcytometry, JC-1 staining was adopted to test mitochondrial membrane potential (△?_m), and cellular structural protein changes were studied with CFDA-SE staining. RESULTS: By 1?10~(-6) mol/L DEX stimulation, the apoptotic rate was 51.25%?5.51% and had significantly difference from control group (12.03%?2.00%); the necrotic rate in DEX group was 30.25%?3.67% and also had significantly difference from control group (10.11%?1.11%, P
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AIM: To study the influence of status of stimulator cells on activation of responder T cells in mixed lymphocyte reaction (MLR), so as to provide some basis for clinical transplantation. METHODS: Stimulator cells were pretreated differently before mixed lymphocyte culture (MLC) to change their functional status, fluorescence conjugated antibodies and flow cytometry were used to detect expression of CD69 by responder T cells at several different time points. RESULTS: The expression percentages of CD69 by responder T cells in MLCa group (stimulator cells were pre-activated) were significantly higher than those in MLC group (stimulator cells were not pre-activated) at 24, 48 and 72 hours of culture, respectively (5 21%?0 24% vs 1 98%?0 33%, 29 81%?0 85% vs 20 65%?1 00% and 39 61%?1 62% vs 13 49%?0 60%, P
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AIM:To study the effects of progesterone(P4) on the maturation and immunologic function of dendritic cells(DCs) from human peripheral blood.METHODS:Cultured DCs were treated with P4 at doses of 10-7 mol/L and 10-6 mol/L.The morphologic changes were observed under the scanning electronic microscope.The immunophenotypes of DCs in control and treated groups were analyzed by flow cytometry.IL-10 and IL-12 production in culture supernatant was examined by ELISA assay.The capability of the stimulatory activity of the DCs on allogeneic T cells in mixed reaction was tested by incorporation of [3H]-TdR.RESULTS:Compared with control group,cultured DCs in the presence of P4 displayed less dendritic pseudopod,expressed low levels of MHC-II,CD40,CD80 and CD86,and exhibited weakly activity in stimulating the proliferation of allogeneic T cells.Increase in IL-10 production and decrease in IL-12 production were observed.CONCLUSION:P4 exerts negative effect on the maturation and immunologic function in dendritic cells from human peripheral blood.
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AIM: To confirm that CD4~+CD25~+ regulatory T cells don't have an instinctive defection in IL-2 secretion, and to have an insight into the maturation state of CD4~+CD25~+ T cells in cord blood. METHODS: CD4~+CD25~+ and CD4~+CD25~- T cells were purified from cord blood of term infants (CB) and adult peripheral blood (PB) by autoMACS, and stimulated with PDB plus ionomycin. After 45 hours of culture, cells were detected for expression of CD69 and CD25 by flow cytometry, and the supernatants were measured for 7 kinds of cytokines by Luminex. RESULTS: CD4~+CD25~+ T cells from both CB and PB proliferated comparably with CD4~+CD25~- T cells when stimulated with PDB plus ionomycin. After 45 hours of culture, however, the CD4~+CD25~+ T cells underwent a tendency of cell death. Expression of CD25 was further upregulated when CD25~+ cells were activated. Under stimulation of PDB plus ionomycin, both CD4~+CD25~+ and CD4~+CD25~- T cells in PB secreted high levels of IFN-?, IL-2 and TNF-?, with CD25~+ cells secreted much higher level of IL-5, IL-4 and IL-10 than those in CD25~- cells; CD4~+CD25~+ and CD4~+CD25~- T cells in CB also secreted high level of IL-2 and TNF-? but much lower level of IFN-? than those in PB, and no secretion of IL-5, IL-4 and IL-10 was observed. CONCLUSION: CD4~+CD25~+ regulatory T cells don't have an instinctive defection in IL-2 secretion, otherwise there may be a different TCR signaling pattern in CD4~+CD25~+ T cells from traditional T cells. The CD4~+CD25~+ T cells in cord blood have not fully matured in function.
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AIM: To study the changes of mitochondrial membrane potential(△?m) and mitochondrial mass in apoptosis of Jurkat cells induced by camptothecin(CPT).METHODS: Jurkat cells were treated with CPT.Annexin V-FITC/propidium iodine(PI) double stainig was used to detected early stage of apoptosis and PI staining for analyzing the cell cycle.Jurkat cells were stained by annexin V-PE/DiOC_6(3) to detect changes of △?m.The mitochondrial mass was measured by cytometry with NAO staining.RESULTS: 6 h after treated with 10 ?mol/L CPT,the rate of early apoptotic cells(22.59?1.04)% had significantly difference compared with control group(3.93?0.73)%(P0.05).Apoptotic peak appeared obviously after treated with CPT,the percentage of late apoptotic cells(13.58?0.97)% had distinctly difference compared with control group(3.18?0.51)%(P
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AIM: To study the effects of ultraviolet (UV) on mitochondrial functions and apoptosis in HaCaT cells.METHODS: After irradiation by UV at low dose(UVA 2 J/cm~2,UVB 10 mJ/cm~2) and high dose(UVA 6 J/cm~2,UVB(30 mJ/cm~2),) HaCaT cells were cultured for 15 hours.Flow cytometry was used to measure mitochondrial membrane potential,mitochondrial mass and apoptotic rate.Annexin V-FITC/PI staining of apoptotic cells was analyzed by laser confocal microscopy.RESULTS: After UV irradiation,cell proportion with low mitochondrial membrane potential increased with irradiation doses.The proportion of control group,low dose group and high dose group were 7.94%?1.02%,25.87%?4.55% and 39.27%?5.32%,respectively.Cells proportion with low mitochondrial mass increased with irradiation doses.The proportion of control group,low dose group and high dose group were 15.19%?1.58%,40.36%?4.41% and 68.79%?5.46%,respectively.The hypodiploid peaks of DNA content analysis represented the apoptotic rate of HaCaT cells.The apoptotic rate of control group,low dose group and high dose group were 1.82%?0.51%,30.16%?5.47% and 58.49%?5.98%,respectively.To analyze the cells apoptosis by staining with annexin V-FITC and PI,the results were consistent with those of DNA content analysis.Cells in control group showed almost no positive staining cells.Single annexin V-FITC positive cells in low dose group and double positive cells in high dose group were predominant,respectively.CONCLUSION: UV irradiation induces HaCaT cell mitochondrial depolarization,as well as mitochondrial mass loss.These changes are related to cell apoptosis.
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AIM: To address whether the analysis of CD45~+CD86~+ cells isolated from para-aortic lymph nodes (PLNs) is valuable in assessment of the status of local immunity at the feto-maternal interface. METHODS: CBA/J?DBA/2, virgin CBA/J, and CBA/J?BALB/c mice were used as an abortion-prone model (group A), non-pregnant controls (group N), and fertile controls (group F), respectively. The percentage of CD45~+CD86~+ cell in the CD45~+ cell group (CD45~+CD86~+ percentage for short) and the absolute number of these cells were determined with flow cytometry (FCM), using mononuclear cells isolated from PLNs collected on day 5.5, 9.5, and 13.5 of gestation, respectively, and mononuclear cells from placentas on day 13.5 of gestation. To clarify the identity of these CD86~+ cells, FCM was also performed with CD3, CD19 and DX5 as markers for T cells, B cells, and NK cells, respectively. RESULTS: Both resorption rate and absolute number of resorption were significantly higher in group A (29.3%, 1.8?1.0) than those in group F (4.8%, 0.3?0.5, P
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AIM: To explore the inhibitory effect of oxymatrine (OMT) on the allergic contact dermatitis (ACD) stimulated by dinitrofluorobenzene (DNFB) and its effects on the proliferation of the lymphocytes. METHODS: ① An ACD mouse model was established by stimulation with DNFB, and then the mice were injected intraperitoneally with different dosages of OMT, PBS and hydrocortisone (HCT) respectively, the swelling degree of their auricles was examined. ② Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) dye and flow cytometer were used to examine the fluorescence intensity changes of lymphocytes stimulated by polyclonal stimulator ConA and OMT. RESULTS: ① compared with PBS group, OMT possessed the strong inhibitory effect on the ACD caused by DNFB in a dose-dependent manner, and its inhibitory effect was equivalent to the HCT of the same dosage with fewer side effects. ② In vitro experiments proved that OMT (500, 125 and 31 mg/L) had the ability to restrain the proliferation of lymphocytes of mouse. CONCLUSION: OMT possesses an inhibitory effect on the ACD induced by DNFB, and OMT is a kind of immunosuppressor.
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0.05) compared with control group at 1 h and 3 h; while ~FL 1 in DEX group at 5 h (660.91?72.95) was significant lower (P
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AIM: To investigate the effect of enhanced green fluorescence protein (EGFP) gene transfection on the cell cycle distribution of primary cultured human chondrocytes in order to establish a tracking method of cultured human nasoseptal chondrocytes. METHODS: pEGFP-N1 plasmid was amplified in E.coli, and purified by high purity kit. Primary cultured human chondrocytes,which were initially obtained from the nasoseptal cartilage, were cultured in vitro and transferred with pEGFP-N1 by means of electroporation with Amaxa nucleofector device. Transfering process and transient expression were evaluated by laser scanning confocal microscope (LSCM), the transfer efficiency and the cell cycle distribution were evaluated by flow cytometry. RESULTS: There was significant expression of EGFP at 24 h after transferring. The transfection efficiency of pEGFP-N1 into primary cultured human chondrocytes reached 35 37% at 48 h. It didn't affect the process of cell adherance and had no effect on the cell cycle distribution. CONCLUSION: Primary cultured human chondrocytes, which were transfected with pEGFP, are alive in vitro, and the transferring process doesn't affect the cell cycle distribution. These results suggest that pEGFP-N1 is an ideal transient expression vector for primary cultured human chondrocytes and it might be a well tracer in construction tissue engineered cartilage.
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AIM: To investigate the effect of cycloheximide on the T cells activation by mitogen in vitro with CD69 expression as activation marker for the application of this drug clinically. METHODS:Lymphocytes were isolated from lymphoid nodes of C57BL/6 mouse. The cells were preincubated with cycloheximide(CHX), 5% serum containing CHX respectively for an hour, then further incubated with polyclonal activators (Con A or PDB). Harvesting the cells after whole incubation for 24 h, we estimated the expression rates of CD69 on T cells by flow cytometry following two-color immunofluorescent staining. RESULTS: The expression rates of CD69 on the T cells preincubated with CHX, serum containing CHX after the stimulation in response to Con A or PDB all showed significant difference with the expression rates of control group, respectively ( P
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AIM: To study the effect of VEGF on extracellular H2O2 production in HUVECs and the role of H2O2 in the VEGF-induced proliferation. METHODS: HUVECs was stimulated with 500 ?g/L VEGF. Products of extracellular H2O2 was detected by H2DCFDA staining. MTT method was used to value the influences of 3?106 U/L catalase and 5-20 mmol/L H2O2 to VEGF function. RESULTS: After treatment for 15 min with VEGF, HUVECs appeared fluorescence, and continued to become stronger, peaked at 45 min then decreased. HUVECs, which was treated simultaneity with VEGF and 3?106 U/L catalase, only appeared very faint fluorescence. The proliferation of HUVECs by VEGF was restrained when treated with 3?106 U/L catalase. The extrinsic H2O2 at concentration of 5-10 mmol/L promoted the proliferation of HUVECs but inhibited the proliferation effect of VEGF on HUVECs (P
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AIM: To study the effect of supernatants from cord blood mononuclear cells (CBMC) and peripheral blood mononuclear cells (PBMC) on infection of HIV-1 in vitro, and develop effectively soluble factors for human acquired immunodeficiency syndrome (AIDS) treatment. METHODS: Different supernatants from CBMC and PBMC activated by PHA for 5 hours and 12 hours were added to cell culture systems between HIV-1ⅢB/H9 cells labeled by calcein-AM and MT-2 cells, then to count the fusion under a reverted fluorescent microscope after 2 hours, respectively, and different soluble factors in supernatants were detected by Luminex 100~ TM . RESULTS: These supernatants from CBMC and PBMC activated by PHA for 5 hours and 12 hours inhibited the formation of fusion, and there is no difference between supernatants collected in CBMC and PBMC at same time point. However, the supernatants collected in 5 hours were more effective to inhibit the formation of fusion than those in 12 hours (P
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AIM: To explore the characteristics of T cell activation and regulatory T cells derived from murine Peyer's patches through comparative studies on Peyer's patches, mesenteric lymph nodes and inguinal lymph nodes. METHODS: Signal cell suspendsions were prepared from murine mesenteric lymph nodes (MLNs), the Peyer's patches (PPs) and inguinal lymph nodes (ILNs), respectively. The percentage of cell subpopulations such as CD3+ T cells, CD3+CD4+ helper T cells and regulatory T cells (Treg, CD4+CD25+) were analyzed. Lymphocytes were activated by polyclonal stimulators such as concanavalin (Con A), phorbol 12, 13-dibutyrate (PDB) only, and PDB plus ionomycin (Ion). The expression of CD69 (the early marker of CD3+ T cell activation) was measured by FACS. RESULTS: A lower ratio of CD3+ T cells was seen in PPs than those in MLNs and ILNs. The ratios of CD3+ CD4+ T cells to CD3+ T cells in PPs, MLNs and ILNs were almost the same. A higher rate of Treg was seen in CD4+ T cells from the PPs as compared with those from MLNs and ILNs. A higher percentage of activated CD3+ T cells derived from the PPs cultured without polyclonal stimulators were detected as compared to MLNs and ILNs, while lower responsiveness of CD3+ T cells from the PPs stimulated by Con A was seen as compared with those from MLNs and ILNs. CONCLUSIONS: The lower rate of CD3+ T cells as well as higher rate of Treg in PPs was due to its desensitization. The higher rate of basic activated state in CD3+ T cells from the PPs indicated that the T cells were activated by enteric antigens in physiological conditions. The lower responsiveness of activation to some polyclonal stimulators probably reveals that the T cells are in a state of anergy. All the characteristics mentioned above contribute to prevent pathological inflammations and maintain tolerance to enteric antigens such as food proteins and commensal bacteria but simultaneously retain proper immune responses to pathogenic microbes.