The Expression Of GSK-3β And β-catenin In Canonical Wnt Signalling From Human Dendritic Cells

Objective:Dendritic cell(DC) can activate immune reaction and induce immune tolerance. The immune function was closely dependent on its maturation status. Immature DC has functions such as uptaking, processing of antigens,but can not provide costimulatory signals, therefore induce immune tolerance,While mature DC with strong antigen presenting function,can active immune response. It was demonstrated that Wnt signalling pathway can regulate the growth of immune cells,which may directly affect the biological function of immune cells. In this study,to investigate the expression of GSK-3βandβ-catenin in dendritic cells,DC was induced from human peripheral blood mononuclear cells in vitro. The morphology,phenotype and functions of cells were analysed. Methods:1. Cells Culture:Enrichment of human peripheral blood leukocytes (buffy coat) from healthy adults was isolated using differential centrifugation and Lymphocyte Separation Medium. The intermediate buffy layer containing PBMC was extracted, and seeded into 25cm2 Flask and then incubated for 3 h in a humidified chamber at 37℃and 5% CO2. After incubation,non-adherent cells were removed,and the adherent cells cultured in fresh medium with GM-CSF and IL-4 for six days. TNF-αwas added on the 6th day for 24 h to induce maturation and obtain immature dendritic cells (iDC) and mature dendritic cells (mDC) on the 5th day and the 7th day respectively.2. morphology,phenotype and functional tests:Morphological features of DC were observed by microscope and electronic microscope. The immunological phenotype of CD1a, HLA-DR, CD83, CD86, CD40, CD80 on DC surface were tested by flow cytometry.Their functional changes were observed by mixed lymphocyte reaction (MLR).3.We examined the expression Profiles of GSK-3βandβ-catenin mRNA by real time PCR.4.Western-blot was used to detect the expression of GSK-3βandβ-catenin in protein level. Results:1.The suspending cells displayed typical morphological features of DC,such as anomalouse, bigger body, numerous dendrites and cascading plica,being observed by both micscope and electronic microscope.2. The mDC show enhanced expression of CD1a,HLA-DR,CD83,CD86,CD40,CD80. And it was promoted the proliferation of non-sensitized T lymphocyte proliferation in MLR than of iDC.3. The expression level of GSK-3βmRNA in iDC was higher than in mDC (P<0.05),and The expression of P-catenin mRNA in mDC was also increased compared to iDC (P<0.05).4. The protein level of GSK-3βin iDC was higher than in mDC,while the level ofβ-catenin protein in mDC was increased compared with that in iDC (P <0.001). Conclusion:1. It was feasible to induce DC from PBMC in enrichment of human peripheral blood leukocytes(buffy coat) by culturation with rhGM-CSF,rhIL-4 and rhTNF-α. We can obtain DC in larger quantities and high purity.2. The Cells exhibited typical morphological characteristics of DC and can manifested different phenotypes and function between iDC and mDC.3. The different expression of GSK-3βandβ-catenin in iDC and mDC was related to the maturation status of DC,which may regulate the mechanism of DC maturation.