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

1.人外周血中内皮祖细胞的分离我们用抗CD34抗体偶联的磁珠从人外周血中分离出CD34阳性的单核细胞。将分离的细胞接种于包被或不包被纤维连接蛋白的塑料平板上。我们使用含有内皮细胞生长因子的培养液。培养48h后，培养板上出现梭形贴壁细胞。当这些梭形细胞与CD34阴性的单个核细胞混合时，它们的增殖和分化范围更广。免疫荧光染色CD34阳性，并掺入Dil标记的血管内皮细胞等乙酰化低密度脂蛋白。体内实验(1)2×10~(-6)人结肠癌细胞皮下接种免疫缺陷小鼠。小鼠尾静脉注射BCECF-AM标记的人内皮祖细胞。接种后1周切除皮下肿瘤，在荧光显微镜下观察肿瘤冰冻切片。但未检测到BCECF-AM阳性细胞。(2)皮下移植瘤小鼠尾静脉注射更多的EPC后，皮下肿瘤中未检测到BCECF-AM标记的人EPC。活体显微镜也没有检测到皮下肿瘤周围内皮祖细胞的聚集。对其他肿瘤细胞系的皮下肿瘤模型也进行了实验，但皮下肿瘤中未见内皮祖细胞积聚。综上所述，体外可分离培养内皮祖细胞，但体内小鼠肿瘤模型中未见内皮祖细胞积聚。这些结果表明，到目前为止，内皮祖细胞应用于抗癌基因治疗是不可能的。

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.