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1.
AIM: To investigate the effects of transforming growth factor β1 (TGF-β1) on murine-derived dendritic cells (DC). METHODS: Murine bone marrow cells were cultured with GM-CSF and TGF-β1 to develop TGF β-DC. Then they were stimulated by lipopolysaccharide (LPS). Their phenotypes were assessed by flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using BrdU ELISA method. IL-12 p70 protein was detected by ELISA and the expressions of Toll like receptor 4 (TLR4) on DCs were measured by semi-quantitative RT-PCR and FCM. RESULTS: Compared to immature DC (imDC) cultured with GM-CSF alone, the expressions of CD80, CD86, I-Ab and CD40 in TGF β-DC were lower. The TGF β-DC was resistant to maturation by LPS. Maturation resistance was evident from a failure to up-regulate CMs, to stimulate larger T cell proliferation and to increase secretion of IL-12 p70. Down-regulation of TLR4 expression on TGF β-DC was also found. CONCLUSION: TGF-β1 inhibits the expression of co-stimulatory molecules on DC. It is resistant to maturation stimulus (LPS) and might be linked with TLR4 down-regulation.  相似文献   

2.
AIM: Recently,it is widely accepted that atherosclerosis (AS) is an auto-immune related disease and the oxidized-low density lipoprotein (ox-LDL) is the most important AS-related antigen.In order to prevent immune injuries in AS and find new strategies to prevent AS,the immune tolerance of T cells to ox-LDL in vitro was induced in this study.METHODS: Human monocytes were separated from peripheral blood to induce dendritic cells (DCs).DCs were treated with LPS (30 μg/L),ox-LDL (10 mg/L) and LDL (10 mg/L) for 48 h.Then DCs were mixed with allogenic T lymphocytes to carry out mixed lymphocytes reaction (MLR).CTLA4Ig in different concentrations was added in the MLR of ox-LDL group.MTT method was used to assay the proliferation of T cells and expressed in stimulation index (IS).The CD25 expression and apoptosis of T cells in MLR were tested by flow cytometry.The excretion of IL-2,IFN-γ and IL-4 was assayed by ELISpot method.RESULTS: SI in ox-LDL group was higher than that in LDL group significantly (P<0.05) and CTLA4Ig inhibited the SI in ox-LDL group with dose-dependent effect (P<0.05,P<0.01).CTLA4Ig decreased the CD25 expression (P<0.05,P<0.01) and induced apoptosis of T cells in MLR (P<0.05,P<0.01).CTLA4Ig decreased the ELISpot counts of IL-2 and IFN-γ (P<0.01),while increased that of IL-4 (P<0.05).CONCLUSION: CTLA4Ig induces T cells tolerance to ox-LDL in vitro.CTLA4Ig inhibits T cells activation,promotes T cells apoptosis and Th1/Th2 immune deviation,which is the important mechanism in it′s induction of tolerance.  相似文献   

3.
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4.
AIM: To study the influence of stimulation by LPS and CD40 ligandization in vitro on the TLR4-MD2 expression and IL-12 production in dendritic cells (DCs) modified by sCD40 gene and provide the experimental clues of inducing dornor-specific immune tolerance.METHODS: Plasmid pEGFP-N1/sCD40 and pEGFP-N1 was transfected into DC2.4 cell line with lipofectamine.After 6 h of treatment with LPS and anti-CD40mAb,the expression of TLR4-MD2 on DCs was determined with flow cytometry (FCM) and RT-PCR.IL-12p70 protein was detected by ELISA.RESULTS: LPS treatment of DCs down-regulated surface expression of TLR4-MD2,LPS treatment along with anti-CD40mAb significantly up-regulated TLR4-MD2 surface expression.CD40 ligandization did not affect TLR4-MD2 mRNA expression in DCs but partly increased its low level induced by LPS and markly enhanced IL-12p70 secretion after LPS stimulation.DCs modified by sCD40 gene inhibited the above effect.CONCLUSION: After treatment with LPS and anti-CD40mAb,DCs modified by sCD40 gene down-regulate surface expression of TLR4-MD2 and IL-12p70 secretion decreases significantly,which might be linked with the interruption of TLR4-MD2 transportation from cytoplasm.  相似文献   

5.
AIM:To evaluate whether tolerogenic dendri tic cells (DC) loaded with heat shock protein 60 (HSP60) inhibit the progression of aortic atherosclerotic plaque in hypercholesterolemic apolipoprotein E (Apo- E) -null mice.METHODS:Bone marrow derived DC of the mice were loaded with HSP 60 and co-cultured with rapamycin to generate tolerogenic DC.The tolerogenic DC ,DC loaded only HSP60 and PBS were injected into the ApoE-null mice at 8 weeks of age for three times at a one-week interval.8 weeks after the last injection,aorta were harvested for HE staining and anti-CD4+T cell immunostaining.Resp onses of pleenic cells to HSP60 were also evaluated.RESULTS:Compared with DC,DCHSP60 expressed higher levels of CD86,and stimulated T lymphocytes to proliferation significantly,while the tolerogenic DC expressed lower levels of CD86,and inhibited T lymphocytes to p roliferation.After immunization with different injection,the numbers of CD4+ T cells in plaque were increased significantly in DCHSP60 group vs in PBS g roup (P<0.01).On the other hand,they were reduced significantly in rap-DC HSP60 group vs in PBS group (P<0.01).Plaque areas in the tolerog enic DC group were smaller than that in the PBS group (P<0.01).Stimulated by HSP60,pleenic cells in tolerogenic DC group secreted more IL-10,while in DC HSP60 group more IFN-γ secretion was observed.CONCLUSION:HSP60 specific tolerogenic DC immunization attenuate d the progression of plaque,indicating a new immune therapy for atherosclerosis.  相似文献   

6.
AIM：To investigate the method of directed differentiation dendritic cells (DC) from embryonic stem cells,and to amplify high purity dendritic cells in vitro for immunolgical therapy.METHODS：E14 embryonic stem cell line was generated ES-DC in complete medium supplemented with GM-CSF and IL-3.Flow cytometry was used to determine CD11c,CD80,CD86,MHC-II cell-surface phenotype in immatured ES-DC.Lipopolysaccharide (LPS) was added to induce the ES-DC maturation.The matured ES-DC was harvested 24 hours later to identifying with morphology and transmission electron microscopy.The phenotype of matured ES-DC was analyzed by flow cytometry and compared with the immatured ES-DC.The antigen presenting was evaluated by mixed lymphocyte responses (MLR).RESULTS：The ES-DC had obviously dendritic processes under scanning electron microscope.The immature DCs expressed low level of CD11c (4.33±0.23)%,CD80 (7.62±0.19) %,CD86 (4.77±1.22) % and MHC-II (9.68±0.15) %.The mature DCs expressed higher level of CD11c (47.36±2.68)%,CD80 (74.40±1.47) %,CD86 (29.77±2.00) % and MHC-II (87.56±2.75) %.MLR showed that ES-DCs effectively stimulated lymphocyte proliferation.CONCLUSIONS：These results provide evidence that dendritic cells can be generated from E14 embryonic cells with the stimulations of GM-CSF and IL-3.The differentiated cells expresse high level of CD11c,CD80,CD86,MHC-II and effectively stimulate lymphocytes to proliferate.ES cells may become new origin of DC for immunotherapy.  相似文献   

7.
AIM: To explore the optimal condition of predominant immature CD8α+ dendritic cells(predo-iDCs) preventing T-cell proliferation pulsed by allogeneic antigen. METHODS: Predo-iDCs, induced with GM-CSF +IL-4 +SCF +Flt3L from SPF healthy C57BL/6 murine bone marrow cells, were pulsed by different doses (0, 2.5, 5, 10 and 20 mg/L)of allogeneic murine splenocyte antigen. Syngeneic T-cells were co-incubated with Ag-pulsed DCs (DCs/T=1∶ 1, 2∶ 1, 4∶ 1) and the T-cell proliferation was measured by MTT. The secretion of cytokines (IFN-γ and IL-10) in the co-incubated supernatants was detected by ELISA. The effect of prevention of T-cell proliferation generated by murine predo-iDCs pulsed by allogeneic antigen was detected. The control derived from mature dendritic cells(mDCs)induced by GM-CSF +IL-4 +TNF-α. RESULTS: The effect of Ag-pulsed predo-iDCs for stimulating T-cell proliferation was the slightest in predo-iDCs/T 1∶ 1 group, compared with that in predo-iDCs/T 2∶ 1 and 4∶ 1 group (P<0.05). The secretion of IFN-γ in mixed lymphocyte reaction(MLR) was significantly lower than the one in mDCs control group, while the secretion of IL-10 was higher than that in control group when low dose of antigen (<2.5 mg/L) was added into MLR. CONCLUSION: Predominant iDCs pulsed by low dose of allogeneic antigen (2.5 mg/L) mixed 1∶ 1 with T-cells is the optimal condition for the prevention of T-cells proliferation.  相似文献   

8.
AIM: To study the dynamic levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and gamma interferon （IFN-γ） produced by human dendritic cells infected with Dengue virus. METHODS: Monocytes isolated from healthy human peripheral blood were incubated in medium with GM-CSF and IL-4 for more than 7 days. DCs were then collected and identified by scanning electron microscope, immunohistochemistry and lymphocytes stimulatory ability. Human dendritic cells （DC) were infected with Dengue-2 virus (DV-2) in vitro， culture supernatants were collected in different time postinfection (6 h, 12 h, 24 h, 48 h and 72 h)， DV antigen in human dendritic cells were demonstrated by an indirect immunofluorescent assay （IFA）, production of TNF-α, IL-6 and IFN-γ in the culture supernatants were evaluated by ELISA. RESULTS: After 7 days, typical dendritic cells could be obtained. Virus antigen were detected in infected DC by IFA. Dengue virus induces TNF-α and IL-6 secretion from DC and does not induce IFN secretion. CONCLUSION: Human dendritic cells are target of dengue virus infection. TNF-α， IL-6 production from DC are increased with DV infection. Dendritic cells may play an important role in DV pathogenicity and immunity.  相似文献   

9.
AIM: To investigate the immunological effect of tumor necrosis factor receptor-associated factor 6 (TRAF6) on the maturation of dendritic cells in vitro.METHODS: The human peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), induced by recombined human granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, were stimulated to mature with TNF-α, LPS or cocktail of cytokines (TNF-α, IL-6, IL-1β and PGE2).The cell surface markers, T-cell stimulatory proliferation capacity and IL-12 p40 production of the DCs were determined by the methods of flow cytometry, ELISA and mixed lymphocyte reation (MLR), respectively.The mRNA expression of TRAF6 was detected by real-time PCR.RESULTS: The expression of TRAF6 was observed in all groups, which in cocktail group was the highest in the DCs with optimum state of maturation.Furthermore, TRAF6 enhanced the production of IL-12 and the ability of T-cell stimulation of mature DCs.CONCLUSION: TRAF6 might play an important role in inducing the maturation of human PBMC-derived DCs.  相似文献   

10.
AIM: To evaluate the ability of dendritic cells (DCs) loaded with tumor lysate to initiate cell-mediated immune responses by stimulating naive T cells, and the efficiency of activated T cells to kill autologous tumor cells in vitro. METHODS: The peripheral blood lymphocytes and monocytes were obtained from the advanced renal cell carcinoma patient by conglutination method. The immature dendritic cells were generated in the presence of interleukin-4(IL-4) and granulocyte/macrophage colony-stimulating factor(GM-CSF) from monocytes of healthy individuals. These cells were pulsed with tumor lysate or not. Induction of tumor-specific cytotoxic T lymphocytes (CTLs) response by mature dendritic cells (mDCs) was evaluated by the CD95(Fas) expression assay through FCM and the cytotoxic assay against autologous human tumor cells.　RESULTS: Human immature dendritic cells and T cells obtained from healthy donors were stimulated with tumor-pulsed dendritic cells. The immature dendritic cells were applied to the cytotoxicity assay against target autologous tumor cells. The CD95(Fas) expression, IFN-γ and TNF-α secreted by the CTLs in tumor lysate-plused DC group were higher than those of other groups. The capacity of the CTLs to kill autologous tumor cells was significantly different(P<0.05). Antigen-specific DCs vaccine can induce T cells activation and proliferation, thus we can obtain higher proportion of tumor specific cytotoxic T cells(CTLs), and enhance the CTLs to secret IFN-γ and TNF-α. CONCLUSION: Our results indicate that monocyte-derived human dendritic cells pulsed with tumor lysate could induce the specific antitumor effect against autologous tumors . This in vitro model offers a new and simple approach to the development of DC+CTL-based immunotherapy.  相似文献   

11.
AIM: To investigate the phenotype and immune activity of dendritic cells using interleukin-18 as intervent.METHODS: Monocytes were isolated from human peripheral blood and induced into DCs with GM-CSF and IL-4. The cellular morphous was observed under inverted microscope. On the 5th day, 3 groups including IL-18 group, TNF-α group and IL-18+TNF-α group were set. IL-18, TNF-α or IL-18+TNF-α was used as intervents respectively to facilitate cell maturity. Supernatants were collected at 24 h, 48 h and 72 h. IL-12 in the supernatant, CD1a, HLA-DR, CD83 and CD86 were analyzed using flow cytometry. DCs of the 3 groups were co-cultured with T cells respectively on the ratio of 1∶〖KG-*2〗100, 1∶〖KG-*2〗50 and 1∶〖KG-*2〗10. T cell proliferation stimulated by DC was determined using MTT method. DCs were co-cultured with T cells on the ratio of 1∶〖KG-*2〗10, and the supernatant were collected at 24 h, 48 h and 72 h. IFN-γ in the supernatant was detected with ELISA method.RESULTS: Induced by GM-CSF and IL-4, then stimulated by IL-18, TNF-α or IL-18+TNF-α, monocytes showed typical morphous of DC. No morphological difference was observed among DCs of the 3 groups. No statistical difference showed in expression level of CD1a, HLA-DR, CD83 and CD86 between IL-18 group and TNF-α group (P>0.05). The positive rates of CD1a and CD83 in IL-18+TNF-α group were higher than those in other 2 groups. The positive rate of HLA-DR in IL-18+TNF-α group was higher than that in IL-18 group. No difference between IL-18 group and TNF-α group in the potency of stimulating T cell proliferation was found, whereas the stimulating potency in IL-18+TNF-α group was higher than that in IL-18 group and TNF-α group. IL-12 in IL-18+TNF-α group at 48 h and 72 h was higher than that in IL-18 group and TNF-α group (P<0.05). However, there was no difference between the latter 2 groups. There was also no difference between IL-18 group and TNF-α group in IFN-γ secretion. IFN-γ in IL-18+TNF-α group was higher than that in IL-18 group and TNF-α group (P<0.05).CONCLUSION: Using IL-18 as intervent, DC expresses high level of surface molecules, secretes high level of IL-12, stimulates T cell proliferating effectively and produces IFN-γ potently. The actions are stronger when used in combination with TNF-α. It suggests that IL-18 may serve as a promoting agent of DC maturity, or combination with TNF-α in DC induction will strengthen the immune activity of DC.  相似文献   

12.
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13.
AIM:To investigate the effect of donor bone marrow derived dentritic cell (DC) treated with B7-1, B7-2 antisense oligonucleotide on mouse heart allografe survival time and its mechanism. METHODS: There were 7 groups of C57BL/10J (B10) mouse bone marrow DCs which were treated by 400 nM antisense oligonucleotide target to B7-1, B7-2 mRNA (AS B7-1/2), B7-1 mismatch oligo control, B7-2 mismatch control(mASB7-1/2), lipofectamine only and non-treatment, respectively. Each group of DC were named as ASB7-1 DC, ASB7-2 DC, mASB7-1DC, mAS B7-2DC, and Lipo DC, respectively.RESULTS: Flow cytometer results shown that AS B7-1/2 can inhibit B7-1(CD80)and B7-2 (CD86) molecule express on DC surface, while control groups have no effects. To observe their tolerogenicity in mouse cardiac allograft model, B10→C3H heterotopic heart transplantation were performed. Recepients were received 2×106 of DC injection 7 days before transplantation. Results showed that both AS B7-1DC and AS B7-2 DC can prolong mouse cardiac allograft survival time to (18.6±0.89) days and (23.67±10.73) days, respectively, compared with IL-4 DC .Two mismatch control groups can slightly prolong while oligo DC has no effect. For understanding its mechanism, each group of DC was used as stimulator to stimulated C3H spleen T cell. Results suggested that AS B7-1DC and AS B7-2 DC had less allo-stimulate function, including MLR and generation CTL and IL-2 production than IL-4 DC but control groups have no effect.CONCLUSION: Donor bone marrow derived DC treated with AS B7-1 oligo and AS B7-2 oligo expressed lower level of CD80 and CD86, respectively. These cells can induce allogeneic T cells anergy in vitro and markedly prolong mouse heart allograft survival time in vivo.  相似文献   

14.
AIM: To investigate the effect of cytomegalovirus (CMV) infection on the immunological functions of dendritic cells (DC) derived from monocytes. METHODS: RT-PCR assay was used to detect the mRNA expression of CMV immediate early antigen (IEA) and glyceraldehyde phosphate dehydrogenase (GAPDH) genes in immature and mature dendritic cell (cmv-imDC, cmv-mDC) infected by 50-folds median tissue culture infective dose (TCID50) of CMV. The expression of early antigen (EA) in cmv-imDC and cmv-mDC was analyzed by indirect immunofluorescence assay. CMV late antigen pp65 was determined by flow cytometry. The allogeneic stimulating capacity of cmv-DC was assayed by mixed lymphocyte reaction (MLR) with BrdU incorporation. RESULTS: The expression of IE mRNA in cmv-mDC was lower than that in cmv-imDCs at 12 h after infection (0.102±0.020 and 0.862±0.124, respectively, P<0.05). EA, primarily localized in nucleus, was found in cmv-imDC (62.32±14.20)% and cmv-mDC (10.78±3.04)% at 24 h (P<0.01). pp65 positive cells in cmv-imDC and cmv-mDC at 72 h were 4.86% and 0.82%, respectively. Compared with untreated mDC, cmv-imDC showed depressed antigen presentation even after stimulated with maturation signal factor LPS (both P<0.05), while cmv-mDC had weaker stimulating capacity only when DC/T cell ratio was 1∶1 (P<0.05). CONCLUSION: CMV efficiently infectes and replicates in imDC. CMV suppresses the antigen presenting capacity of cmv-DC.  相似文献   

15.
AIM: To investigate the effect of uric acid on the maturation and the biological function of murine bone-marrow derived dendritic cells (BMDCs) in vitro.METHODS: BMDCs were cultured with GM-CSF, IL-4, LPS and uric acid. Cell phenotypes were analyzed by flow cytometry. MTT was used to detect the effect of uric acid on the function of BMDCs in stimulating the proliferation of T cells. IL-12 released by BMDCs was also detected. RESULTS: BMDCs were cultured and identified. Uric acid at concentrations of 200 mg/L and 400 mg/L increased the expression of the molecules (CD11c, CD83, CD86, IA/IE) on BMDCs surface and the IL-12 level in the culture supernatants (P<0.05), promoted the proliferation of T cells at the T: DC rate 5∶1, 10∶1, 20∶1 (P<0.05). However, uric acid at concentration of 70 mg/L had no effect on above molecule expression (P>0.05), no effect on T cell proliferation with BMDCs (P>0.05) was observed. CONCLUSION: Uric acid promotes the differentiation, maturation, the expression of co-stimulatory molecules, the IL-12 production in BMDCs and enhances the ability of BMDCs to stimulate the proliferation of T cell in a specical dose range.  相似文献   

16.
AIM: To examine whether tolerogenic dendritic cells (DC) loaded with heat shock protein 60 (HSP60) could restore endothelial function in hypercholesterolemic apolipoprotein E (apoE)-null mice.METHODS: Bone marrow derived DC of the mice was loaded with HSP60 and co-cultured with rapamycin to generate tolerogenic DC.The tolerogenic DC, DC loaded only with HSP60 (DChsp) and saline were injected into the apoE-null mice at 6 weeks of age for two times at a one-week interval.C57BL/6 mice at the same age were taken as normal control two weeks after the last injection.Aorta was harvested for ex vivo vascular ring tension test.Immune parameters were also analyzed in vitro and in vivo.RESULTS: Compared with the non loaded DC, HSP60 pulsed DC expressed higher levels of CD86, and stimulated T lymphocytes to proliferation significantly, while the tolerogenic DC expressed lower levels of CD86, and inhibited T lymphocytes to proliferation.After immunization with different injection, Ach-induced relaxation was reduced significantly in DChsp group compared with saline group (P<0.01).Treatment of mice with tolerogenic DC restored endothelium-dependent dilation in a dose-dependent manner (P<0.01).The improvement in endothelial function was associated with a reduction in T cell response to HSP60.CONCLUSION: Our results indicate a rapid improvement in endothelial function with HSP60 tolerogenic DC immunization, and suggest that this immune therapy has significant vasculoprotective effects.  相似文献   

17.
AIM: To investigate the effect of artemisinin on lipopolysaccharide(LPS)-induced intestinal epithelial barrier damage in IEC-6 cells and its molecular mechanism. METHODS: Cultured IEC-6 cells were divided to 5 groups: control group, LPS(100 mg/L) group and LPS+Artemisinin(30, 50 and 100 μmol/L) groups. The cytotoxicity was detected by MTT assay. The releases of TNF-α, IL-1β and IL-6 in the IEC-6 cells were measured by ELISA. The transepithelial electrical resistance(TER) was detected by electrical resistance tester, and the horseradish peroxidase(HRP) flux permeability were analyzed by a microplate reader. The expression of tight junction proteins, ZO-1, claudin-1 and occludin, and the expression of TLR4/MyD88/NF-κB at mRNA and protein levels were determined by RT-qPCR and Western blot. RESULTS: Artemisinin alone(up to 100 μmol/L) or in combination with LPS(100 mg/L) was not toxic to IEC-6 cells. Compared with control group, the releases of TNF-α, IL-1β and IL-6 in the culture supernatant of IEC-6 cells significantly increased after treatment with LPS. The expression of TLR4/MyD88/NF-κB was activated by LPS. LPS down-regulated the protein expression of ZO-1, claudin-1 and occludin. However, artemisinin treatment decreased the releases of TNF-α, IL-1β and IL-6 in the culture supernatant of IEC-6 cells. The expression of TLR4/MyD88/NF-κB at mRNA and protein levels was gradually reduced after treatment with artemisinin. In addition, artemisinin upregulated the protein expression of ZO-1, claudin-1 and occludin significantly(P<0.01) in a dose-dependent manner. CONCLUSION: Artemisinin attenuates LPS-induced intestinal epithelial barrier damage by inhibiting TLR4/MyD88/NF-κB activation in the IEC-6 cells.  相似文献   

18.
AIM: To investigate the immune function of dendritic cells (DCs) in patients with hepatocellular carcinoma(HCC). METHODS: The DCs were cultured from human peripheral blood mononuclear cells (PBMC) by using GM-CSF and IL-4 for 7 days. Surface molecules CD86, HLA-DR of DCs were detected by flow cytometry. IL-12 production by DCs and IFN-γ production by T cells was measured with ELISA and ELISPOT, respectively. Allogenic mixed lymphocyte reaction was detected by MTT assay. RESULTS: CD86 expression in DCs in HCC patients were markedly lower than that in health control (91.7％ vs 83.5%, P<0.05). IL-12 production by DCs had no significant difference between the HCC patients and the health control ［(324.6±171.0)ng/L vs (436.5±142.7)ng/L, P>0.05］. However, after stimulated DCs with LPS, IL-12 production in HCC patients was significantly lower than that in health control ［(478.6±142.7)ng/L vs (630.0±151.9)ng/L, P<0.05］. IFN-γ production by T cells and T cell proliferation index (PI) in the HCC patients were all significantly lower than those in health control ［(IFN-γ: 133.4±51.2)103U/L vs (183.0±60.2)103U/L, P<0.05; PI: 2.3±0.7 vs 3.5±0.8, P<0.01］. CTL killing activity assay indicated that DC-induced CTL killing activity in HCC group was significantly lower than that in health group when the E/T ratio was 10∶1 and 20∶1, respectively. CONCLUSION: The immune function of DCs and T cells in the patients with HCC are significantly decreased. The CTL killing activity of HCC group is also markedly decreased.  相似文献   

19.
AIM:To investigate the effects of invariant natural killer T-cells (iNKT cells) from ovalbumin (OVA)-induced asthmatic mice combined with OVA on the phenotypic and functional characteristics of bone marrow-derived dendritic cells (BMDCs) in vitro. METHODS:The BMDCs from wild-type (WT) BLAB/c mice were co-cultured with purified iNKT cells from WT mice immunized and challenged with OVA in the presence of 100 mg/L OVA (iNKT cells plus OVA group) or PBS (iNKT cells plus PBS group) for 20 h, and were also cultured with 50 mg/L LPS (LPS group), 100 mg/L OVA (OVA group) or PBS (PBS group) for 20 h. The expression of MHC-Ⅱ, CD40, CD86, and CD80 on the BMDCs was measured by flow cytometric analysis, and the levels of interleukin-12 (IL-12) p70, IL-6, tumor necrosis factor-α (TNF-α) and IL-10 in the culture supernatant were measured by ELISA. Splenic CD4+ T cells from DO11.10 transgenic mice were co-cultured for 48 h with the above mentioned BMDCs, and then the concentrations of IL-4 and interferon-γ (IFN-γ) in culture supernatants were measured by ELISA. RESULTS:The expression of MHC-Ⅱ, CD80, CD86 and CD40, and releases of proinflammatory cytokines by BMDCs in iNKT cells plus OVA group were comparable to those in the LPS group (P>0.05), but significantly higher than those in iNKT cells plus PBS group, OVA group, and PBS group (P<0.05 or P<0.01). The concentration of IL-4 in culture supernatants from BMDCs in iNKT cells plus OVA group co-cultured with DO11.10 CD4+ T cells was similar to that in LPS group (P>0.05), but markedly higher than that in iNKT cells plus PBS group, OVA group, and PBS group (P<0.01). CONCLUSION:Bone marrow-derived dendritic cells undergo immunogenic maturation upon interaction with iNKT cells in the presence of OVA.  相似文献   

20.
AIM: To study the expressing variation of TNF-α and IFN-γ mRNA in mouse splenocytes induced by H22 tumor cells derived heat shock protein gp96-peptide complexes in vitro,and to observe the morphologic change of H22 tumor cells which treated with the culture supernatant.METHODS: H22 tumor cells derived HSP gp96 was obtained by the techniques for protein extraction and purification and was identified by Western blotting method.The expression values of TNF-α and IFN-γ mRNA in spleen lymphocytes were detected by semi-quantitative RT-PCR.The morphologic changes of H22 tumor cells induced by the culture supernatant were observed by laser scanning confocal microscopy (LSCM) and transmission electron microscope (TEM).RESULTS: Purified heat shock protein gp96 was identified by Western blotting.The expression value of TNF-α and IFN-γ mRNA in activated spleen lymphocytes induced by gp96-peptide complexes was higher than that in control groups (P<0.05).The morphologic change of apoptosis of H22 tumor cells,which treated by the culture supernatant of experimental group was observed with LSCM and TEM.CONCLUSION: Heat shock protein gp96-peptide complexes increase the expression value of TNF-α and IFN-γ mRNA in spleen lymphocytes of mouse in vitro.Besides,apoptosis of H22 cells is induced by immunologic active material secreted by activated splenocytes.  相似文献   

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