Study on the change of migration ability of dendritic cells induced by intermittent hypoxia and the mechanism of its pathway / 重庆医学

Objective By simulating the intermittent hypoxia(IH ) environment of human obstructive sleep apnea syndrome (OSAS) ,to reveal the effect of IH on migratory ability of human peripheral blood derived dendritic cells(DCs) ,and through the in‐tervention of RelB ,p38 expression in order to explore the possible mechanism of the change of DCs migration ability induced by IH . Methods DCs were divided into RelB ,p38 siRNA interfering and non interfering plasmid group before cultivation .The environment of hypoxia was created by a intermittent hypoxia cabin ,among them ,oxygen concentration was 0 .5% ,1 .5% ,5 .0% ,10 .0% ,hypox‐ia/reoxygenation time ratio was set as 1∶1 ,1∶3 ,1∶5 and 1∶9 ,while sustained oxygen was supplied to the contrast at a normal concentration of 21 .0% .The content of RelB and p38 was tested by Western blotting after culture in vitro ,migration ability of DCs was detected by invasion chamber .Results Compared with normoxia ,DCs under IH tended to have declined migratory ability , which was confirmed to be correlated with the average oxygen partial pressure level under IH .IH could promote the expression of RelB and p38 in DCs ,while the migratory ability of DCs was not reversed after intervening the expression of RelB and p38 .Conclu‐sion IH in vitro could cause a decline in migratory ability of DCs ,which may not be induced by activation of RelB or p38 in DCs .

Effect of RelB-silenced BMDC pulsed with Tα_(146～162) on immunoreaction of T cells primed with TAChR / 中南大学学报(医学版)

Objective To investigate whether RelB-silenced bone marrow-derived dendritic cells (BMDC) pulsed with torpedo acetylcholine receptor (TAChR) immuno-dominant peptide Tα_(146～162) can induce tolerance in T cells primed with TAChR. Methods Recombinant lentivirus that produced RelB siRNA and control lentivirus were prepared and used to infect BMDCs. The infected BMDCs were stimulated with LPS,and the resulting cells were designated as DC-siRelB or DC-control, respectively. The mRNA and protein expression of RelB were examined by quantitative real-time PCR and Western blot. Cell surface markers of DC were evaluated by flow cytometry. IL-12 in the supernatant was detected by ELISA. Mice were randomly divided into 6 groups: A1, A2, A3,K1, K2, and K3. On day 0, group A1, A2, and A3 were primed with TAChR in CFA and group K1, K2 and K3 were primed with KLH+CFA. On day 7, group A2 and K2 were injected with Tα_(146～162) pulsed DC-siRelB, group A3 and K3 were injected with Tα_(146～162) pulsed DC-control, while A1 and K1 group received PBS at the same time. On day 14, lymphocyte proliferative response of the 4 groups were measured. Results Recombinant lentivirus including RelBshRNA genes was successfully constructed. RelB siRNA knocked down RelB expression in BMDCs obviously. Compared with DC-control, DC-siRelB expressed a significantly lower level of CD80, CD86, and MHC class II on their surface, producing lower level of IL-12. Compared with group A1 and A3, lymphocyte proliferative response to TAChR of A2 group was suppressed significantly (P<0.05). No different lymphocyte proliferative responses to KLH and ConA were seen in group A1, A2 and A3 (P>0.05). No different lymphocyte proliferative responses were seen in group K1, K2 and K3 (P>0.05). Conclusion Lentiviral-mediated RelB-silenced BMDCs are maturation resistant and can induce antigen-specific tolerance in TAChR primed C57BL/6 mice,which provides a basis for further study of their therapeutic potential in myasthenia gravis.

Biological effects of RelB gene silencing on the dendritic cells derived from bone marrow / 解放军医学杂志

Objective To study the biological effects of nucleic transcript factor NF-?B/RelB gene silencing on bone marrow derived dendritic cells(BMDCs).Methods For constructing a new type of DCs,RNAi RelB DC,mouse BMDCs were cultured through IL-4 and GM-CSF cytokine inducing system and were purified with CD11c positive immunomicrobeads by flow cytometer,and RelB gene was silenced with PCR expression cassettes.The RelB RNA interference effect in DCs was proved at both mRNA and protein levels.The biological characteristics of RNAi RelB DCs were investigated by means of cell morphology,cell surface markers and mixed lymphocyte reaction ability.The cell microstructure was observed using scanning electron microscopy and transmission electron microscopy.The cell surface markers,such as MHC-Ⅱ,CD86 and CD40,were detected by flow cytometer.The mixed lymphocyte reaction ability,as well as the ability of DCs in stimulating allogeneic T cells proliferation,was detected by 3H-TdR incorporation method.Results RNAi RelB DCs seemed to have fewer and shorter spikes and many folds under scanning electron microscopy.Many phagocytic vacuoles were observed in the cytoplasm under transmission electron microscopy.DCs surface molecules,MHC-Ⅱ,CD86 and CD40,were expressed at a relatively lower level compared with the control DCs.This RNAi RelB DCs also showed weak allogeneic T cells stimulating ability in vitro in MLR.Conclusion The dendritic cells derived from bone marrow are characterized the biological features and immunological function of immature DC with silencing of RelB gene.This kind of RNAi RelB DCs may be used to induce immunotolerance as a novel tolerogenic DCs.

Construction and identification of RNAi lentiviral vector on murine RelB / 解放军医学杂志

Objective To construct a lentiviral expression vector of murine RelB for RNAi,and then to effectively silence the RelB gene expression of murine bone marrow derived dendritic cells for constructing the bone marrow tolerogenic dendritic cell,in order to provide an experimental foundation for a novel clinical therapy of autoimmune disease.Methods RelB shRNA sequence of mouse was designed by on-line designer software on Corp.Invitrogen,after synthesis and annealing,double strand oligonucleotides(dsoligoes)were cloned into the pENTRTM/U6 plasmid,then after sequencing,a positive clone was further subcloned into pLenti6/BLOCK-iTTM-DEST vector.It was then transformed into stb13 competent cell,and after sequencing,293FT cell line was transfected by above positive recombined plasmid and lentiviral packing materials.It was incubated for 48 to 72 h in a 37℃,5% CO2 incubator.Culture supernatant was harvested and stored at-80℃,then the virus titer was determined by serial dilution assay.Results It was showed from sequencing figures that all the pENTRTM/U6-RelB-shRNA plasmids were positive clone vector,and this positive recombinant vector was recombined with pLenti6/BLOCK-iTTM/U6-DEST vector.It was then transformed into stb13 competent cell and screen positive clone by ampicillin,and resequenced by the use of primer forward U6 primer.The results also showed that recombinant lentiviral vector was positive clone.Viral particle was packaged with other packaging material mediated by lipofectamine 2000 in 293FT cell line.Cultural supernatant was collected and stored at-80℃,and lentiviral particle titer was determined by serial dilution assay with 6?105/transduced unit.Conclusion Lentiviral shRNA expression vector of murine RelB gene for RNAi was successfully constructed.It might be a rational procedure to make tolerogenic DC,and then to develop a DC vaccine and DC-based immunotherapy for autoimmune diseases.