| Dendritic cells (DCs) are the most potent antigen presenting cells (APCs) that prime naive T lymphocytes and active antigen specific cytotoxic T lymphocytes(CTLs), which makes DC a promising tools for immunotherapy of cancer .Althought DCs are widely distributed in tissues in vivo,the isolation of DCs from blood and other tissues has been difficult in view of their scarcity. Recently, different methods have been developed to obtain DCs in vitro from different progenitors.However, the mechanism by which cytokines promote the DCs differentiation in vitro remains unknown. Clarification of DCs differentiation mechanism should be criticle for effectively obtaining abundant DCs Studies have showed the growth, differentiation and maturation of hematopoietic lineage induced by cytokines are always related with intercellular signal transduction, and JAK-STAT pathway maybe the main pathway. Whether JAK-STAT pathway taking part in and it's effect on the DCs differentiation from CD34+ cells are not clear. Against this background, we sought to isolate CD34+ hematopoietic stem cells from human umbilical cord blood and cultured in GM-CSF,FL,SCF,TNF-αand IL-4 for two weeks to obtain large amount of DCs with the antigen capturing and processing capacity in vitro. Total cellular and phosphorylated JAK2 and STAT5 at different time point during DC differentiation were evaluated by western blotting .The main results are described as follows:By Ficoll and Vario-MACS,CD34+ hematopoietic stem cells, can be rapidly and efficiently enriched to a purity of 99.08% . CD34+ hematopoietic stem cells from huam cord blood can differentiate into large amount of DCs when cultured in presence of GM-CSF,FL,SCF,TNF-α and IL-4 in vitro for two weeks. The cells expanded from human cord blood CD34+ cells have typical dendritic morphology and highly expressed differential antigens on cell surface .Nevertheless,the induced DCs had the capacity to stimulate proliferation of allogenetic T1. lymphocytes.Therefore, these cells differentiated from cord blood CD34+ cells are DCs with special function.4. The amount of JAK2 protein was similar at d0,d7 and d14 without GM-CSF stimulation.But the tyrosine phosphorylation pattern after GM-CSF stimulation differed markedly.With cells differentiating to DCs, the amount of tyrosine phosphorylated JAK2 increased markedly.5. Both total cellular and tyrosine phosphorylated STAT5 expression increased markedly during DC differentation.6. JAK2 protein at d14 were actived quickly with GM-CSF stimulation. Maximal tyrosine phosphorylated STAT5 expression was later than JAK2. Conclusion:1. Positive seleced with Vario-MACS,CD34+ hematopoietic stem cells, can be rapidly and efficiently enriched to high purity. This method may facilitate further generation of high purity of DCs. 2.CD34+ hematopoietic stem cells from huam cord blood can differentiate into large amount of active DCs when cultured in presence of GM-CSF,FL,SCF,TNF-α and IL-4 in vitro for two weeks. These DCs have typical dendritic morphology and highly expressed differential antigens.Nevertheless,the induced DCs had the capacity to stimulate proliferation of allogenetic T lymphocytes.3.The recently purified CD34+ cells from huam cord blood were hyporesponsiveness to GM-CSF.2. JAK2 phosphorylation and STAT5 activation induced by GM-CSF are upregulated during the differentiation of CD34+ stem cells into DC,from which we can conclude that JAK2-STAT5 pathway may take part in the signal mechanism of DCs differentiation...
|
PDF Full Text Request