【作者】 宫安静；

【导师】 孟庆海；

【作者基本信息】 青岛大学 ， 神经外科， 2004， 硕士

【摘要】 目的：研究负载肿瘤抗原的树突状细胞（DCs）活化的特异性细胞毒性T淋巴细胞（CTLs）对神经胶质瘤细胞的体外杀伤效应，探讨其用于临床治疗的可行性。方法：体外原代培养胶质瘤细胞，冻融法获取胶质瘤细胞抗原，联合应用粒细胞／巨噬细胞集落刺激因子、白细胞介素-4和肿瘤坏死因子-α等，对人外周血单核细胞进行体外诱导来获取DCs，负载肿瘤抗原后，激活自体T淋巴细胞，制备特异性CTLs，MTT法检测对胶质瘤细胞的体外杀伤效应。结果：自诱导第2天起，倒置显微镜下观察到部分细胞体积增大，形态由圆形变得不规则，成多形性，可见驼峰样或细刺状胞浆突起。DCs培养第10天，流式细胞仪检测DCs高表达CD_1a、CD₈₀、CD₈₃、CD₈₆、HLA-DR。同种异体混合淋巴细胞反应显示少量DCs即可明显刺激同种异体淋巴细胞增殖且DCs与淋巴细胞比例在1：10时淋巴细胞增殖最明显。负载胶质瘤抗原的DCs激活的CTLs对胶质瘤细胞的杀伤作用显著高于对K562细胞的杀伤作用（P＜0.01），并且杀伤活性随着效靶比的增加而增加。负载胶质瘤抗原的DCs激活的CTLs对胶质瘤的杀伤活性显著高于未经抗原致敏的DCs刺激的CTLs对胶质瘤的杀伤作用（P＜0.01）。上述两组CTLs对K562细胞杀伤作用类似（P＞0.05）。未经抗原致敏的DCs刺激的CTLs对胶质瘤细胞和K562细胞杀伤无显著差别（P＞0.05） 结论：联合应用细胞因子rhGM-CSF、rhIL-4、rhTNF-α可从人外周血中诱导出典型的树突状细胞，将DCs负载胶质瘤抗原后，激活的CTLs在体外对胶质瘤细胞能产生高效而特异的杀伤作用，为临床应用DCs瘤苗奠定基础。更多还原

【Abstract】 Objective To investigate the antitumor efficiency of the special cytotoxic T lymphocytes activated by dendritic cells loaded with glioma antigens in vitro and the feasibility of immunotherapy of glioma with dendritic cells for clinical application. Method Glioma cells were prepared by primary culture in vitro and tumour antigens were extracted by thawing and freezing. Peripheral blood monocytes were cultured to produce DCs with recombinant human granulocyte-macrophage colony - stimulating factor, recombinant human interleukin-4, recombinant human tumor necrosis factor- . After pulsed in vitro with glioma lysates, DCs were used to induce T lymphocytes into cytotoxic T lymphocytes for glioma cells. The cytotoxicity of CTLs to patient’ s glioma cells was assayed by MTT. Result After culter for two days, cells began to increase in size and be charactered with dendritic morphology. After 10 days, more CDla , CD80, CD83, CD86, HLA-DR were detected by flow cytometer. Dendritic cells stimulated lymphocytes to increase greatly in Allo-MLRs. It was obvious when the proportion was 1 to 10. The CTLs could kill glioma cells markedly in vitro and the killing rate of glioma cells was more higher than that of K562 (P<0.01) .The ratio increased with the improvement of the proportion of effector cells to target cells. The CTLs were more effective to glioma than the CTLs cultured without glioma antigens (P<0.01) . Tow kinds of CTLs had similar effect to K562 (P>.05) .Conclusion The antigen specific CTLs killed glioma cells effectively and specifically, which might play a great role in clinical therapy.更多还原