嵌合锚定T细胞致癌胚抗原阳性胃癌细胞的凋亡效应  

T lymphocytes with chimeric receptor induce carcinoembryonic antigen-positive specific gastric carcinoma cells apoptosis

作　　者：徐宏勇[1] 徐立[2] 高建宏[1] 李开宗[3] 窦科峰[3] 

机构地区：[1]解放军第451医院普外科,西安710054 [2]第四军医大学西京医院消化病研究所 [3]解放军第451医院肝胆外科,西安10054

出　　处：《中华医学杂志》2007年第15期1053-1057,共5页National Medical Journal of China

基　　金：国家自然科学基金(39900068);西安市科技攻关计划项目基金资助(GG05168)

摘　　要：目的探讨 CEA 靶向性嵌合锚定 T 细胞的制备方法,并检测其活化功能及诱导 CEA阳性胃癌细胞的杀伤活性。方法分离外周血单个核细胞(PBMC),然后用免疫磁珠分离得到 CD^+_8T 细胞。将重组真核表达载体抗-CEA-scFv-CD3 zeta-pcDNA3.0采用脂质体转染上述 T 细胞以制备CEA 靶向性的嵌合锚定 T 细胞;用流式细胞仪检测其 CD69的表达,以检测嵌合锚定 T 细胞活化功能;将嵌合锚定 T 细胞与胃癌细胞 HGC-27、SGC-7901共培养,检测后者膜联蛋白 V 的表达,分析嵌合锚定 T 细胞致胃癌细胞的凋亡效应。结果将已制备的 CEA 靶向性嵌合锚定 T 细胞培养12 h、24 h后,检测 T 细胞的活化信号 CD69表达率分别为40.5%±3.4%、48.3%±2.8%,证明嵌合锚定 T 细胞具备活化功能。检测与嵌合锚定 T 细胞共培养的胃癌细胞 HGC-27、SGC-7901,凋亡率分别为47.8%±4.2%、18.7%±2.8%,两者之间有统计学意义(P<0.05)。结论 CEA 靶向性嵌合锚定T细胞可特异性识别 CEA 阳性胃癌细胞并促使后者凋亡,这种治疗策略将为胃癌的临床诊治提供一种有希望的途径。Objective To develop a method to generate T lymphocytes that identify tumor specific carcinoembryonic antigen （CEA）, and induce anti-tumor response such as apoptosis. Methods Peripheral blood samples were collected from 10 healthy persons and peripheral blood mononuclear cells（PBMCs）were isolated. 1, then CD8^＋ T cells were isolated from the PBMCs by magnetic activated cell sorting （MACS） using magnetic beads-conjugated anti-CD8 monoclonal antibodies. Then the recombinant vector anti-CEA-scFv-CD3zeta-pcDNA3.0 was transfected into the CD8^＋ T cells by lipofectamine 2000 and T lymphocytes with chimeric receptor were generated and cultivated. The T lymphocytes＇ activation was assessed by detecting the expression of CD69, an early signal of T cell activation. Human gastric carcinoma cells of the lines HGC-27 （ CEA^＋ ） and SGC-7901 （ CEA^- ） were cocultivated with the T lymphocytes with chimeric receptor. Flow cytometry （FCM） with annexin V-FITC/PI double staining was used to detect the expressions of annexin V, a signal of cell apoptosis, so as to observe the apoptosis of the gastric carcinoma cells. T cells activated by phytohemagglutinin （PHA） were used as positive controls, and T cells transfected with blank vector pcDNA3. 0 were used as negative controls. Results T lymphocytes with chimeric receptor directed towards CEA^＋ gastric carcinoma cells were generated. 12 and 24 hours after the co-culture of these T cells and HCG-27 cells, the CD69 expression rates were 40. 5% ± 3.4% and 48. 3% ± 2. 8% respectively. However, the CD69 expression rates of the HGC-27 and SGC-7901 cells transfected with the T lymphocytes transfected with blank vector were both 0%. FCM showed that the apoptotic rates of the CEA^＋ gastric tumor HGC-27 cells was 47.8% ± 4. 2%, significantly higher than that of the CEA^- gastric tumor SGC-7901 cells （18.7% ± 2. 8%, P 〈 0. 05 ） . Conclusion T lymphocytes with chimeric receptor targeting CEA specifically identify CEA positive gastric carcinoma cell

关 键 词：胃肿瘤 T淋巴细胞 癌胚抗原 

分 类 号：R735.2[医药卫生—肿瘤]