负载HPV18E7基因脐带血树突状细胞的体外抗食管癌研究

目的制备人脐带血CD34^＋造血干细胞来源的负载HPV18E7基因的树突状细胞（DC）疫苗，并观察其在体外对人食管癌细胞的杀伤活性。方法采用微磁珠分选系统（MiniMACS）从脐带血单个核细胞中分离CD34干细胞，以重组人粒细胞．巨噬细胞集落刺激因子（rh—GM-CSF）和重组人肿瘤坏死因子-α（rhTNF-α）各200ng／ml和100U／ml诱导其向DC分化，采用阳离子脂质体DMRIE-C介导HPV18E7基因进入DC，制备DC疫苗。倒置相差显微镜下观察DC的生长情况和形态变化；流式细胞仪检测疫苗表面目的基因E7蛋白的表达情况；MTY法检测DC疫苗致敏的同种异体外周血T淋巴细胞在体外对表达HPV18E7的食管癌细胞EC-109的杀伤活性。结果脐带血CD34^＋干细胞在细胞因子诱导下，细胞体积增大，形状由圆形变得不规则形，细胞数量增多，形成细胞集落。经过14d的培养，大部分的细胞从集落上脱落下来成为成熟的DC，具有典型的树枝状突起。E7蛋白的阳性表达率为47．5％，说明基因转染成功。DC疫苗致敏的淋巴细胞在体外对EC-109细胞的杀伤活性明显强于未转基因DC致敏的淋巴细胞组、T淋巴细胞组和对照组（P〈0．01），且随效靶比的增高而增强（P〈0．05）。结论人脐带血干细胞在细胞因子的诱导下能扩增分化为DC，经肿瘤相关病毒抗原基因转染获得的DC疫苗，高表达目的基因蛋白，并能显著增强淋巴细胞对相应人食管癌细胞的体外杀伤效应。

In vitro anti-esophageal carcinoma immune response induced by cord blood-derived dendritic cell loaded with HPV18E7 gene

Objective To develop a dendritic cell-based cancer vaccine,which derived from human cord blood CD34^＋ hemopoietie stem cell and then loaded with HPV18E7 gene,study its eytotoxic activity on human esophageal carcinoma cells in vitro. Methods CD34^＋ stem cells were isolated from human cord blood mononuelear ceils by Mini MACS, and then stimulated into DC with recombined human granuloeytemacrophage colony-stimulating factor （rhGM-CSF,200 ng/ml） and recombined human tumor necrosis factor- α（rhTNF-α, 100 U/ml）. After 12 days euhuring, HPV18E7 gene were transfeeted into DC inducing by cationic lipesome （DMRIE-C） to prepare DC vaccine. The change in cell morphology and ~ell number during the differentiation process of DC was observed under inverted phase contrast microscope. The expression of E7 protein on DC vaccine was detected with flow cytometry. In vitro,methyl thiazolyl tetrazolium （MTT） assay was used to detect the cytotoxie activity of allogeneie T lymphocytes induced by DC vaccine against esophageal carcinoma cell line EC-109. Results Under the stimulation of cytokines, the CD34^＋ ceils became large and irregular in shape, the cell number increased and formed cell clones. 14 days later, cells became matured and shed from the clones, which stretched out pronounced typical long dendritic processes.The expression rate of E7 protein was 47.5% ,which suggested efficient gene transfer. The cytotoxic activity of DC vaccine primed T lymphocytes were significantly higher than that of untransfected DC primed lymphocytes,T cell group and control group （ P 〈 0. 01 ）, and the cytotoxicity enhanced with the increasing stimulation by DC vaccine （P 〈 0. 05）. Conclusions CD34^＋ stem cells from human cord blood can expand and differentiate into DC under the stimulation of cytokines. The expression of aim gene protein were extremely high in DC vaccine loaded with tumor associated virus antigen gene, and the DC vaccine can significantly enhance the cytotoxic activity of T lymphocytes against corm- sponding esophageal carcinoma cells in vitro.