| Part I CTLA4Ig Gene Were Transducted into Dendritic Cells by AdenovirusObjective:To construct CTLA4Ig gene modified dendritic cells.Methods:The mouse bone marrow cells were isolated and cultured in complete medium with GM-CSF and IL-4for7days. Cell growth and morphological changes were observed under optical micro scope. The phenotype and the maturation of dendritic cells were analyzed by flow cytometry, examining the expression of CD11c, MHC Ⅱ, CD80and CD86. The cells were incubated with recombinant adenovirus (AdCTLA4Ig, AdGFP)and OVA for48hour, then target gene expression were observed under the fluorescence microscopy, transfection efficiency were determined by flow cytometry.Results:1. Mouse bone marrow-derived cells showed characteristic dendritic morphology of dendritic cells and formed cell colony on the day7.2. The cell phenotype determined by flow cytometry showed that64.01%of cells were positive for CD11c,33.49%of the cells were positive for CD11c+CD80+cells,14.65%of the cells were positive for CD11c+CD86+cells,21.53%of the cells were positive for CD11c+MHCⅡ+cells.3. Incubated with recombinant adenovirus and OVA for48hours, green fluorescence in cells was observed under a fluorescence microscope. Flow cytometry analysis showed AdCTLA4Ig transfection rate was41.29%.4. Incubated with recombinant adenovirus and OVA for48hours,42.65%of the cells were CD11c+CD80+cells,22.30%of the cells were CD11c+CD86+cells,32.46%of the cells were CD11c+MHC Ⅱ+cells.Conclusion:Constructing CTLA4Ig gene modified dendritic cells successfully. Part Ⅱ The Functional Studies of CTLA4Ig Gene-modified Dendritic Cells in vitroObjective:To study the function of CTLA4Ig gene-modified dendritic cells in vitro.Methods:The mouse bone marrow-derived DCs were isolated and cultivated. The cultured dendritic cells for7days were divided into four groups:DC group, DC+OVA group, the other two groups were added1μl(1.5×108VP/ml) AdGFP or AdCTLA4Ig and OVA. All groups were cultured for48hours, then supernatant and cells were separated. The levels of IL-10and IL-12in supernatant were measured by ELISA. Cells were used to perform mixed lymphocyte reaction with peripheral blood T-lymphocytes of BALB/c mice.Results:1. CTLA4Ig gene transfected in dendritic cells promoted IL-10secretion (P<0.05).2. CTLA4Ig gene transfected in dendritic cells, reduced IL-12secretion (P<0.05).3. CTLA4Ig gene transfected in dendritic cells decreased the dendritic cells’ability to promote T lymphocyte proliferation (P<0.05).Conclusion:CTLA4Ig gene modified dendritic cells can promote the expression of IL-10, reduce the expression of IL-12and supress T cell proliferation. Part III CTLA4Ig Gene-modified Dendritic Cells Transfused To Mice In Neonatal Period Suppress Adult Airway Allergic InflammationObjective:To study the impact of CTLA4Ig gene-modified dendritic cells transfused to mice in neonatal period on the airway inflammation of adult OVA-sensitized/challenged mice, and to explore its pathogenesis.Methods:Use OVA-sensitized/challenged mice by intraperitoneal injection and inhalation methods to establish asthma model. The CTLA4Ig gene-modified dendritic cells were given to the newborn BALB/c mice, OVA-sensitized/challenged the mice in the adulthood. Establish the AdGFP intervention group with the same method. In addition, establish the control group and asthma model group. The manifestation of OVA challenged mice in each group, BALF eosinophils and lung pathological changes were observed to study the impact of CTLA4Ig gene-modified dendritic cells on airway inflammation. The level of IgE, IL-4, IL-10and INF-γ were detected by ELISA methods.Results:1. Asthma model were established successfully by OVA-sensitized/challenged mice.2. CTLA4Ig gene-modified dendritic cells given in neonatal period significantly improved the symptoms of asthma in adulthood, reduced eosinophils in BALF and improved the airway inflammation in biopsy.3. CTLA4Ig gene-modified dendritic cells reduced IgE levels in serum and BALF during the onset of adulthood asthma.4. CTLA4Ig gene-modified dendritic cells reduced IL-4level in serum and BALF during the onset of adulthood asthma.5. CTLA4Ig gene-modified dendritic cells increased IL-10level in serum during the onset of adulthood asthma.6. CTLA4Ig gene-modified dendritic cells had no impact on level of INF-y in serum and BALF during the onset of adulthood asthma.Conclusion:CTLA4Ig gene-modified dendritic cells given in neonatal period suppress the airway inflammation of OVA-sensitized/challenged mice in adulthood, elevate the serum levels of IL-10, reduce the secretion of Th2cytokines, has no impact on the secretion of Thl cytokines. In conclusion, this study provides a new idea to prevent and contol asthma. |
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