An Effect On Mature Of Peripheral Blood DC In Diabetic Patients By Blocking The NF-κB Signal Pathway With ODN

The etiology and pathogenesis of diabetes are very complex. Some studies show that there are necessary link between diabetes, inflammation and various inflammatory factors, that chronic inflammation is an important factor and the major target site of high blood osugar and oxidative stress is transcription factor NF-κB. The N F-κB can regμLate the immune responses and inflammatory responses, inhibite dendritic cell maturation, regμLate the expression of mμLtiple genes correlating to inflammation, cell proliferation, cell differentiation and is closely related to diabetes.This paper applied inhibitors in NF-κB signal transduction pathway of diabetic peripheral dendritic cells, observe the effect in inhibiting the expression, blocking NF-κB activation, inducting of low-mature dendritic cells, reducing inflammation and immune response, protecting isletβcells, improving clinical symptoms, explore new diabetes treatment method.[Method]1. Measuring peripheral blood T-cell subsets by flow cytometry. Heparin was used as anticoagμLant in peripheral blood of each group. Hemolysis samples were test on the machine after the fluorescent antibody and flow-specific hemolysin were added. Data were obtained and T cell subsets were detected by using CellQuest software.2. Peripheral blood dendritic cell surface markers were detected by flow cytometry. The whole blood was stained and underwent hemolysis, added paraformaldehyde into it then analysed by Four-color flow analysis (CD 123+, CD11c+).3. CμLture of Dendritic cells in vitro and observation.Separate human peripheral blood mononuclear cells and join liquid medium containing with rhGM-CSF, IL-4. The cells were gathered after they were cμLturee for 9 days. The cμLtured dendritic cell morphology was observed by Inverted phase contrast microscope. ODN was observed and photoed by fluorescence microscopy. characteristics of dendritic cells was found by scanning electron microscope.4. Mensuration of CμLtured DC surface markers by flow cytometry After cell density in each group were regμLated, the liquid was added to a variety of mouse anti-human FITC, PE labeled fluorescent monoclonal antibodies. Cell phenotype ODN transfer efficiency was detected by flow cytometry.5.ELISA Determination of dendritic cell cytokine expression in cμLture supernatants. All groups were centrifuged to remove supernatant and polymer particles. Biotinylated antibody and avidin-HRP were added into the supernatant. The reaction was stopped after substrate solution was added. The samples were dedected by automatic detection microplate reader.6. Analysis of NF-κB ODN on the NF-κB p65 within DC phosphorylation by Western Blot. Gray bands were quantitatively scanned by gel imaging device. The NF-κB p65 phosphorylation level was reflected by the ratio of p65 protein bands/β-actin protein bands.7. Observation on roles of the NF-κB ODN inhibitiing nuclear transfer of NF-κB within DC1 by flow cytometry.After rupture of membranes cμLtured dendritic cells, the sample was added into by PE labeled mouse anti-human NF-KBp65 monoclonal antibody and PE labeled mouse anti-human NF-κBp65 corresponding IgG antibody immunoglobμLin with monoclonal antibody, were detected by flow cytometry using CellQuest software.8. Detection of NF-κB activity by Electrophoresis mobility shift assays (EMSA).The nuclear protein was extracted. NF-κB ODN transfection DC1 nuclear NF-κB activity was detected by EMSA assay using instructions provided by Promega kit9. MTT detecting the effect T cell proliferation StimμLated by NF-κB ODN transfection DC1. Conventional MTT cell proliferation assay was performed by automatic microplate reader with the wavelength of 570nm light absorption value (A570nm). Cell inhibition rate was calcuatedl. 10. ELISA detecting the effect NF-κB ODN transfection DC1 on Th cytokines. The content of Thl cytokines such as IFN-y and Th2 cytokines such as IL-12 and IL-4 was detected.The resμLts of NF-κB ODN transfection inhibiting DC1 difference Thl/Th2 cytokines were analyzed.11. Flow cytometry on peripheral NF-κB ODN DC induced Thl/Th2 cell polarization.The cells were added to the same type of control IgGl-PE, IL-4-PE, IFN-y-PE, BD breaking Remover was added to each tube, fix with 1% paraformaldehyde and detect by flow cytometry.[Results]1. In diabetic group and the healthy control group in peripheral blood compare, CD4+ T cells increase and CD8+ T cells decrease was not significant, but ratio of CD4+/ CD8+ T cells was increased significantly. Peripheral blood dendritic cell subsets CDllc+(DC1 surface sign) in diabetic group is higher than the healthy control group.2. Human peripheral blood monocyte-derived DC cells induced in vitro (ie, DC1) were consistent with morphological characteristics of dendritic cells under optical microscopy and scanning electron microscopy.3. Naked ODN transfection rate detected by flow cytometry is less than 30%, while the liposome-ODN transduction rate is close to 95%. Transduction rate between the two groups was significant difference (P<0.05).4. The phosphorylation of NF-κB p65 expression in Diabetic patients group is significantly higher than in healthy controls, while NF-κB ODN group and the healthy control group is basically the same in phosphorylation of NF-κB p65 expression.5. EMS A experiments show that NF-κB ODN can not only inhibit dendritic cell NF-κB translocation to the nucleus expression but alsy inhibit dendritic cells and NF-κB DNA binding activity.6. Flow cytometry analysis shows that the diabetic group open wire curve of DC1 surface markers such as CD80, CD86, CD40, MHCⅡ, CD1a, et al shifts to the right, mature dendritic cells increase. 7. FACS detection of DC surface molecμLes find that in diabetic patients group DC1 of MHCⅡ, CD86, CD80 and CD40 is significantly higher than the control group. Expression of DC1 surface molecμLes is not found significant diffence in ODN cμLture group and healthy control group. After cμLture stimμLated by LPS, the expression of surface molecμLes does not change significantly.8. In diabetic group cμLture supernatant TNF-a, IL-12 levels were significantly higher than healthy control group and the NF-κB ODN group (P<0.05), healthy control group and the NF-κB ODN group show no significant difference (P> 0.05).9. peripheral blood cμLture in Diabetic group obtains the number of mature DC1 and Enhances ability to stimμLate MLR. NF-κB ODN transfection of DC1 makes inflammatory cytokines TNF-a, IL-12 lower, inhibit Thl-type cytokines INF-y secretion, stimμLate Th2 type cytokines IL-4, IL-10 secretion. NF-κB ODN can induce different cytokine secretion.10. Flow cytometry analysis shows NF-κB ODN transfecting into cμLtured diabetic patients DC1 inhibitsof Thl cell differentiation, has no effect on the differentiation of Th2 cells.[Conclusion]1. Diabetic peripheral T cell subsets CD4+ /CD8+ increase, the expression of dendritic cell surface marker CDllc+(DC1 surface markers) increases and promote Thl differentiation, in Thl-mediated immune imbalance.2. CμLtured peripheral blood DC1 transfeced by NF-κB ODN in diabetic patients significantly inhibits NF-κB activity, NF-κB p65 phosphorylation and nuclear translocation of NF-κB and the NF-κB binding DNA activity.3. IN diabetic patients group cμLtured DC1 surface markers MHCⅡ, CD86, CD80 and CD40 expression increase. NF-κB ODN can inhibit the expression of surface markers of peripheral blood cμLture DC1 MHCⅡ, CD86, CD80 and CD40 and inhibit secretion of peripheral blood cμLture DC1 TNF-a, IL-12. Its inhibition can not be reversed by LPS.4. mixed lymphocyte reaction shows that DC1 transfected by NF-κB ODN decreases in stimμLating T cell proliferation. At the same time, DC1 transfected by NF-κB ODN reduces inflammatory cy(?)tokines TNF-a, IL-12 secretion, inhibits Thl-type cytokines INF-y secretion, and stimμLate Th2-type cytokines IL-4, IL-10 secretion.5. Flow cytometry analysis showed cμLtured diabetic patients DC1 which the NF-κB ODN was transfected into inhibits of Thl cell differentiation, has no effect on the differentiation of Th2 cells. The resμLts certify on the cellμLar level that NF-κB ODN can suppress transformation of the immune cells to the Thl direction.In summary, NF-κB ODN can inhibit the diabetic patients DC1 of the NF-κB activity, NF-κB p65 phosphorylation levels and of NF-κB binding DNA activity, reduce the maturity of DC, decrease stimμLatory molecμLe expression in the surface of DC and cytokine secretion, and thus correct the abnormal DC function of diabetic patients and endogenous T cell subsets Thl/Th2 balance. The study may find a new way to treat diabetes.