The Establishment Of EA.hy926 Expressing GM-CSF And IL-4 Stably To Induce The Differentiation Of PBMC Into DC

Dendritic cells are unique in the ability to recruit naive immune effector cells and to stimulate memory effector cells.DCs for research and clinical applications are usually obtained by culturing PBMC-derived monocytes in a cocktail of cytokines for about 1 week. However, Because it has been suggested that these cytokine-derived DCs may be deficient in some important immunological functions and might not accurately represent antigen presenting cell(APC) populations found under normal conditions in vivo, there is an interest in developing methods that permit the derivation of DCs in a more physiologically relevant manner in vitro.several laboratories have worked to develop in vitro systems which more closely recapitulate the cell interactions and signaling pathways that trigger monocyte to DC differentiation in vivo.For instance,the groups of Muller and Randolph pioneered the development of a tissue construct consisting of primary HUVEC grown atop a collagen support matrix that promotes the differentiation of blood monocytes into APCs which resembled DCs by phenotype and function. Its complexity makes it impractical for widespread use. Schanen described a simple, cost-effective and convenient Transwell-based culture method for the endothelial cell-mediated differentiation of human DCs from bloodmonocytes. This system generates APCs with a frequency and purity comparable to more traditional DC populations derived in vitro, but did so in only 2 days, in the absence of exogenous factors and without the need for a cumbersome matrix to support the growth of HUVECs. However,it’s complicate to isolate HUVEC from human umbilical cord and culture in vivro.For this reason,we choose EA.hy926 which is obtained by fusion of HUVEC and A549 to study .GM-CSF and IL-4 play an important role in modulating proliferation, differentiation and survival of dendritic cells. The EA.hy926 cell line expressing GM-CSF and IL-4 stably has been established by lentiviral system. The cell line we contrasted will be of great significance to the culture of DC.This paper can be divided into two part :(1)The establishment of feeder cells ;(2) PBMC are cocultured with feeder cells we constructed in the first part .In this paper , The recombinant lentiviral plasmid carrying human GM-CSF and IL-4 succesfully constucted ; then infectious lentiviral was obtained and can efficiently infected the EA.hy926 cells.the ability to express GM-CSF and IL-4 in the infected EA.hy926 was identified by RT-PCR, Real-time quantitative PCR , Western bloting and ELISA.,and the results told us that the infected EA.hy926 can express GM-CSF and IL-4 in a high level and can be cultured in a long term.In this paper we determined the action time and concentration of MMCwhich is used to the establishment of feeder cells .In the last,the DCs were obtained by PBMC coculturing with feeder cells.1.the establishment of feeder cells with expressing GM-CSF and IL-4 The gene GM-CSF-T2A-IL4 was obtained by SOE PCR and then insert into lentiviral plasmid pBPLV. The recombinant lentiviral plasmid was identified by Double enzyme digestion and then transfected into 293FT cells with lentiviral packaging plasmids.the efficiency of transfection was 90% and .the efficiency of infection was 94%. The infected EA.hy926 can be cultured in vitro in a long term. GM-CSF and IL-4 expression at mRNA level of infected EA.hy926 cells are 24 and 9946-fold for the EA.hy926 not infected by the lentivirus, and then at the protein level are 145 and 23-fold.2. PBMC are cocultured with feeder cells we constructed in the first part 10μg/ml MMC was used to establish feeder cells.The feeder cells could keep the ability to express protein ,but no generation; CD14+ monocyte was isolated by CD14+ magnetic beads and then concultued with feeder cells ; DCs from coculture was identified by flow cytometry.The result showed that the feeder cells we constructed can promote the differentiation of monocyte into DC.The coculture-derived DCs expressed costimulatory molecules and HLA-DR in a hign level and express CD1a、CD83 .