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目的研究多种恶性胶质瘤相关抗原表位肽激活的树突状细胞(GDC)致敏的细胞毒性T淋巴细胞(GDC-CTL)对人脑胶质瘤细胞系U87的体外细胞毒活性,以及其对BALB/c nude裸小鼠神经胶质瘤模型的体内抗肿瘤作用。方法制备多种恶性胶质瘤相关抗原致敏的GDC和GDC-CTL细胞,用流式细胞仪对细胞表型进行分析。设立不同的效靶比(5∶1,10∶1,20∶1),用CCK8法测定GDC-CTL细胞体外对U87细胞的杀伤率,用酶联免疫吸附实验(ELISA)法检测GDC-CTL与U87细胞共培养48 h后培养上清液中γ干扰素(IFN-γ)水平,设立未经GDC激活的T淋巴细胞为对照组。用BALB/c Nude裸小鼠U87细胞皮下肿瘤模型观察GDC-CTL细胞体内抑瘤作用,实验分为空白对照组、模型组、静脉治疗组和局部治疗组,空白对照组于背部皮下注射0.9%Na Cl,其他3组于背部皮下注射1×107U 87细胞,在肿瘤长径达3 mm后模型组于肿瘤局部注射0.9%Na Cl,静脉治疗组和局部治疗组分别于尾静脉和肿瘤局部注射2×107GDC-CTL,每周注射3次,共2周,注射体积均为0.2 m L。观察4组小鼠的肿瘤体积生长情况,取肿瘤组织进行病理学检测。结果 GDC高表达CD83、CD1a和HLA-DR,表明成功诱导GDC成熟。CD3+T淋巴细胞比例为93.00%,CD3+CD8+T淋巴细胞为69.00%,表明GDC-CTL成功激活。效靶比为5∶1,10∶1,20∶1时:GDC-CTL组和对照组对U87细胞的杀伤率分别为(24.35±1.12)%vs(15.21±0.91)%,(38.57±2.10)%vs(23.35±1.30)%,(59.44±3.79)%vs(35.23±2.33)%;GDC-CTL组和对照组与U87细胞共培养48 h后培养上清液中IFN-γ水平分别为(405.36±27.65)vs(371.11±23.23)pg·m L~(-1),(1509.22±97.16)vs(913.54±48.35)pg·m L~(-1),(2429.57±183.18)vs(1814.97±123.24)pg·m L~(-1),在效靶比为10∶1和20∶1时,2组间差异有统计学意义(P<0.05)。静脉治疗组和局部治疗组对BALB/c nude裸小鼠皮下神经胶质瘤的生长抑制率分别为34.83%和45.37%,与模型组的100.00%比较,差异均有统计学意义(P<0.05),与模型组相比,局部治疗组和静脉治疗组均可见肿瘤细胞显著减少。结论 GDC-CTL是一种高效的免疫细胞,具有较强的体内外抑制胶质瘤生长的作用。
Objective To investigate the cytotoxicity of GDC-CTL sensitized with various malignant glioma-associated epitope peptide-activated dendritic cells (GDC) to human glioma cell line U87 in vitro, And its in vivo anti-tumor effect on a BALB / c nude nude mouse glioma model. Methods GDC and GDC-CTL cells sensitized with various malignant glioma-associated antigens were prepared and their cell phenotypes were analyzed by flow cytometry. The killing ratio of GDC-CTL cells to U87 cells was determined by CCK8 assay, and the effect of GDC-CTL was determined by enzyme linked immunosorbent assay (ELISA) After co-cultured with U87 cells for 48 h, the level of IFN-γ in the culture supernatant was determined, and the T lymphocytes that were not activated by GDC were set as the control group. The inhibition of GDC-CTL cells in vivo was observed by subcutaneous tumor model of BALB / c Nude nude mice. The experiment was divided into blank control group, model group, intravenous treatment group and local treatment group. The blank control group was subcutaneously injected 0.9% Na Cl. The other three groups were subcutaneously injected with 1 × 107U 87 cells in the back. After the tumor diameter reached 3 mm, the model group was injected with 0.9% NaCl locally into the tumor. The intravenous and local treatment groups were injected with tail vein and tumor respectively 2 × 107GDC-CTL, injected 3 times a week for 2 weeks, the injection volume was 0.2 m L. The growth of tumor volume in 4 groups of mice was observed, and the tumor tissues were taken for pathological examination. Results GDC overexpressed CD83, CD1a and HLA-DR, indicating successful induction of GDC maturation. The percentage of CD3 + T lymphocytes was 93.00% and the percentage of CD3 + CD8 + T lymphocytes was 69.00%, indicating that GDC-CTL was successfully activated. The killing rates of U87 cells in GDC-CTL group and control group were (24.35 ± 1.12)% vs (15.21 ± 0.91)% and (38.57 ± 2.10), respectively CTL group and control group, the IFN-γ levels in culture supernatants after 48 h co-culture with U87 cells were (23.35 ± 1.30)% and (59.44 ± 3.79)% vs (35.23 ± 2.33)% (405.36 ± 27.65) vs (371.11 ± 23.23) pg · m L -1, (1509.22 ± 97.16) vs (913.54 ± 48.35) pg · m L -1, (2429.57 ± 183.18) vs (1814.97 ± 123.24) pg · m L ~ (-1). When the target ratios were 10:1 and 20:1, the difference between the two groups was statistically significant (P <0.05). The growth inhibition rates of subcutaneous glioma in BALB / c nude mice were 34.83% and 45.37%, respectively, which were significantly different from those of model group (P <0.05) ), Compared with the model group, the local treatment group and the vein treatment group were significantly reduced tumor cells. Conclusion GDC-CTL is an efficient immune cell with strong inhibition of glioma growth in vitro and in vivo.