消化器癌の増殖・転移に対する血管内皮前駆細胞の関与と遺伝子治療への応用

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1. Isolation of Endothelial Progenitor Cells (EPCs) from Human Peripheral Blood We isolated CD34-positive mononuclear cells from human peripheral blood by using magnetic beads conjugated with an anti-CD34 antibody. These isolated cells were cultured on the plastic plates coated with or without fibronectin. we used the culture medium containing some endothelial cell growth factors. Spindle-shaped adherent cells appeared on the plates 48 hr after the culture. These spindle-shaped cells proliferated and differentiated more extensively when mixed with CD34-negative mononuclear cells. These cells were positive for CD34 by immunofluorescent staining, and incorporated Dil-labeled acetylated low-density lipoprotein such as endothelial cells.2. In Vivo Experiments(1) 2x10^6 of human colon carcinoma cells were subcutaneously inoculated in immunodeficient mice. Human EPCs which were labeled with BCECF-AM were injected into tail vein in the mice. The subcutaneous tumors were excised 1 week after the inoculation and the frozen sections of the tumors were observed by a fluorescent microscopy. BCECF-AM-positive cells, however, were not detected in the samples.(2) Human EPCs labeled with BCECF-AM were not observed in the subcutaneous tumors when more EPCs were subcutaneously injected into tail vein of the mice with subcutaneous tumors. In vivo microscopy did not detect the accumulation of EPCs around the subcutaneous tumors as well. Subcutaneous tumor models of other cancer cell lines were also tested but EPCs were not accumulated in the subcutaneous tumors.Taken together, EPCs could be isolated and cultured in vitro, but EPCs were not accumulated in the mice tumor models in vivo. These results suggest the application of EPCs to gene therapy against cancer is not possible up to the present.