The Research On The Function And The Foundation Of The Structure Of Dendritic Cells

objectivesThis researtch will focus on 3 following aspects: 1) the method of culturing mature and functional dendritic cells (DC), which is identified by typical appearance, standard phentotype expressing CD83, CD86, CD80, HLA-DR, CD11c rich, but Lin-1 negative, as well as by the efficient priming of T cell response i.e., activation and proliferation of T cells, cytokine production, changes in ratios of T cells subsets, etc. 2) Studies on ultrastucrure of DC by inverted phase contrast microscope, scaning electron microscope, transmission electron microscope, atomic force microscope (AFM) and the situation of markers like mature markers (CD83, CD1a), constimulation molecules (CD86, CD80), the origin symbol (CD11c) by confocal microscope to try to find the structure for the function. 3) Observation of morphological alteration of DC and development of Ca2+ in DC during their interaction with T cells, Material and Methods1. MaterialReagents: Mouse mAb to human (FITC-conjugated) CD86, HLA-DR, CD11c, Lin-1, CD4, IgG2a, CD45RA, CD69, Lin-1, Mouse mAb to human (PE-conjugated) CD83, CD86, CD80, CD3, CD123, CD40, CD4, CD25, CD28, CD45RO, IFN-r, IL-4, Mouse mAb to human CD4-APC, CD8PE-Cy5, HLA-DR-percp, and Annexin V-FITC/ PI di-dyed kits for FCM.Equipment: Carbon dioxide incubation box, Invered phase contrast microscope, Flow cytometry (FCM), Scaning electron microscope, Transmission electron microscope, Atomic force microscope (AFM), Confocal microscope, etc,2. Methods1) Using granulocyte/macrophage colony-stimulating factor(GM-CSF) 800u/ml, interleukin 4(IL-4) 5007ml,and tumor necrosis factor a (TNF-a ) 250u/ml, the DC were generated in AIM-V with or without nonadherent cells relatively. The DC were observed by invered phase contrast microscope and photographed every day.2) Flow cytometry analyzed the DC population stained with CD83, CD86, CD80, CD123, CD40, CD11c and CD1a based on HLA-DR positive and Lin-1 negative.3) FCM calculated the changes of CD69 in T cells, as well as CD4/CD3, CD4/CD25, CD45RO/CD45RA, CD4/CD28 sebsets of CD4+T and CD8+T cells.4) Propidium iodide (a nucleic acid dye) stained the DNA of the T cells in presence of RNAase and with DC hibitated by Mitomycin C and then FCM noted the periods DC too.5) To characterize the polarization of T cells, the secretion of IFN- Y and IL-4 by T cells was described by FCM.6) The percentage of DC with apoptpsis was determined with Annexin V-FITCY PI di-dyed kits for FCM.7) Ultrastucture of DC was evaluted at scaning electron microscope, transmission electron microscope to obtain the special structure.8) To further explore the ultrastucture of DC, atomic force microscope performed it at the resolution of nm.9) The situation of CD86 at DC was located with electron microscope immunohistochemical technique.10) population of molecules of CD expressing at DC was further exemed with confocal microscopy, and the action between DC and T cells, processes of phagocytosis and apoptosis, changes of Ca2+ in DC were observed too.Results1. DC showed dramatic changes in cell morphology during culture within 7 days. Many dendritic projections appeared at the adherent cells gradually observed by light microscope; By scaning electron microscope, typical "veiled" DC appeared with abundance of gauffer and the edge of the veil made of graded dendritic projections; all kinds of DC conjected and composed of cluster by the projections.2. The percentages of CD markers were CD123/54.5, CD11c/88.99, CD40/56.65, CD1a/69.36, CD86/92.57, CD80/70.79, CD83/85.91.3. The numbers of T cells expressing CD69 has been increased strikly (CD4+CD69+/19.05%; CD8+CD69+/2l.93%).4. The augment of percentages of CD3+, CD25+, CD45RO+/CD45RA+, CD4+/CD28-and CD8+/CD28- and the decrease of CD28+ and CD45RO-/CD45RA+ in both the CD4+ and the CD8+ T cells could be exemed.5. After priming the T cells response, the ratios of T cells...