Stable Expression Of Gm-csf, Il - 4 Ea. Hy926 Established Cell Lines

Dendritic cells (DCs) are the most potent antigen-presenting cells with a u nique ability to stimulate naive T cells and initiate primary immune response, which phagocytes many kinds of antigen, and expresses lots of MHC, costimul-atory molecules, adhesion molecule, and stimulates cytotoxicitic T cells to kill tumor cells, it is in a central position in the immune response system. DC’s unique immunoregulation makes it becoming the hot study field in immunology, and it has a wild application prospect in the field of the treatment of cells. DCs for research and clinical applications are usually obtained by culturing PB MC-derived monocytes in a cocktail of cytokines for about1week. However, several laboratories have worked to develop in vitro systems which more closel y recapitulate the cell interactions and signaling pathways that trigger monocyte s to DC differentiation in vivo.In this paper, we have sucessfully constructed two recombinant lentiviral e xpression plasmids which seperately carrying the GM-CSF gene and the IL-4g ene, and then established two EA.hy926cell lines which can respectively expre ss GM-CSF and IL-4stably by lentiviral system. The ability to express GM-C SF and IL-4in the two infected cell lines was indentified by RT-PCR、Wester n blot and ELISA, and the results told us that the two infected EA.hy926eel1lines can respectively express GM-CSF and IL-4in a high level and can be cultured in a long term. The two cell lines we contructed will be of great sign ificance to the culture of DC. We also determined the action time and concent ration of MMC which is used to the establishment of feeder cells.In the last, the DCs were obtained by PBMC coculturing with feeder cells.This thesis is composed of two parts as followed:1. The establishment of two recombinant EA.hy926cell lines.The gene GM-CSF and the gene IL-4was obtained by PCR and then ins ert into lentiviral expression plasmid pBPLV. The recombinant lentiviral plasmid was identified by Double enzyme digestion and then transfected into293T eel Is with lentiviral packaging plasmids. The efficiency of transfection was above90%.Then collected the cell culture supernatant, and then high-speed centrifug ation of concentrated virus stock solution, the concentration of the virus infecti on of endothelial cell line EA.hy926cells, the infected cells after expanding cu lture of efficiency of infection is still more than90%, indicating that the target gene can be stable within infected cells and passaged recombinant EA.hy926endothelial cell lines was successfully constructed.2, the determination of the nature of the two restructuring EA.hy926cell1inesWe determined the growth curves of the infected cell lines, the results sho wed that the two infected cells was not affected and both have good proliferati on; we found the target gene expression at the mRNA level by RT-PCR reacti on; ELISA method showed that the infected of IL-4expression of GM-CSF pr otein levels were80times and143times the uninfected cells; by Western blot to detect recombinant cell culture supernatant expression of cytokines IL-4an d GM-CSF; mitomycin C (MMC), inhibition of endothelial cell line growth ex periment to determine the processing time for2.5h, the MMC concentration wa s selected as10μg/ml. The effect of this concentration, the growth of endotheli al cells was inhibited, but to maintain the function of its secretion.