Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiles in dendritic cells from Peyer's patches and epidermal of skin

ABSTRACT: Many studies have shown that the mucous membranes and skin are at the interface with different external environments and face the disparity of pathogenic effects, such as biological agents, chemical or physical environment. This difference may demand distinct immune responses. However, the mechanism to induce the distinct immune responses in mucous and skin is largely unknown. Dendritic cells of mucosa and skin are crucial in the initiation of immune responses, maintenance of self-tolerance and antigens presentation T cells. The different functions between mucosal and epidermal dendritic cells may play an important role in different immune responses. To compare the different gene expression of the mucosal DC and Langerhans cells (LC), we utilized microarrays to investigate different gene expression profiles in mucosal DC isolated from PPs (PDC) and epidermal LC from skin (ELC). 3548 genes were shown to be differentially expressed between PDC and ELC. According to genes annotations, 105 genes may be involved in immunity process. The genes involved in immune process were categorized to five groups related to DC function, including antigen presentation, antigen uptake, cytokines chemokines, and receptors, cell surface molecules and signal transduction. 11 of the highest expressed genes were selected as the candidate genes and reformed by real-time PCR. These 11 selected genes might be suitable candidates to further study the difference of gene expression between mucosal DC and epidermal LC and would be used for design for new vaccine. Beads and FACS sort were used to isolate dendritic cells from Peyer's patches and epidermal of skin from 80 four-week-old female BALB/c mice. Dendritic cells from Peyer's patches were pooled as one sample, and dendritic cells from epidermal of skin were pooled as another sample. RNA isolation, amplification, cDNA labelling, microarray hybridization and analyses were performed according to the Affymetrix manual book.

ORGANISM(S): Mus musculus

SUBMITTER: quan xing

PROVIDER: E-GEOD-15754 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In this study transcriptome profiling of dendritic cell subtypes was performed using various human dendritic cells. Monocyte-derived DC: CD14+ monocytes were obtained from buffy coats by Ficoll - gradient centrifugation and immunomagnetic cell separation using anti-CD14-conjugated microbeads. Monocytes were resuspended into 6-well culture dishes at a density of 1.5 x 10 million cells/ml and cultured in RPMI 1640 supplemented with 10% FBS containing 800 U/ml GM-CSF and 500 U/ml IL-4. Cells were cultured for 5 days and the IL-4 and GM-CSF addition was repeated at day 2. Dermal DC (DDC) and Langerhans Cells (LC) from skin: Human skin specimens were obtained from healthy donors undergoing corrective breast or abdominal plastic surgery after informed consent. Thick slices (3 mm) of skin containing the epidermis and the dermis were cut by use of a dermatome. Slices of skin were cut in pieces of 1 cm2 and incubated with Dispase II for 30â60 min at 37Â°C. The epidermis and dermis were separated with tweezers and washed with PBS. To isolate LC, the epidermal sheets were incubated with PBS with 0.05% trypsin for 10 min at 37Â°C, and a single-cell suspension was prepared by pushing the tissue through 100 Âµm pore nylon cell strainers with a plunger of a 2-ml syringe. Epidermal cell suspension was enriched for LC by density centrifugation over Lymphoprep and CD1a-guided MACS (Miltenyi Biotec). To isolate DDC, the dermis was incubated with PBS containing Dispase and Collagenase A at 37Â°C for 2 h, after which, a single-cell suspension was prepared by pushing through 100 Âµm pore nylon cell strainers with a plunger of a 2-ml syringe. Cell suspension was enriched for DDC by CD1a-guided MACS. Blood Cd1c+ DC: Blood CD1c+ DC were isolated from periferal blood. BDCA1+ (CD1c) DCs were incubated with a lineage-specific PE-labeled Ab mixture, HLA-DR-allophycocyanin, and BDCA1-FITC. Lineage-negative and HLA-DRhigh DCs, positive for BDCA1 were sorted on a FACS and collected in tubes containing 1 ml of 100% FBS. A total of 40,000â100,000 cells was isolated (>98% pure). Dendritic cell subtypes were differentiated in vitro from monocytes and primary dendritic cells were isolated from human skin and blood.

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Gene expression profiles in dendritic cells from Peyer's patches and epidermal of skin

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