左旋-1-甲基色氨酸联合DC疫苗抗乳腺癌免疫效应的研究

发布时间：2018-07-25 11:50

【摘要】：目的:确定小鼠乳腺癌细胞4T1抗原致敏的树突状细胞(DC)疫苗,在体内外抗肿瘤免疫的效应;探讨吲哚-2,3-双加氧酶(IDO)化学抑制剂左旋-1-甲基色氨酸(L-1-MT)联合DC疫苗治疗对荷乳腺癌小鼠肿瘤引流淋巴结(TDLN)中IDO表达水平的抑制程度及其增强荷瘤小鼠肿瘤免疫应答的机制。方法:1、体外培养BALB/c小鼠骨髓来源的DC,用小鼠乳腺癌细胞4T1冻融抗原致敏来制备肿瘤特异性DC疫苗。流式细胞仪检测DC成熟表型;混合淋巴细胞反应(MLR)检测DC刺激T淋巴细胞增殖能力;以4T1为靶细胞,DC致敏的T细胞为效应细胞检测细胞毒性T淋巴细胞(CTL)体外特异性杀瘤效应。2、雌性BALB/c小鼠于第四脂肪垫下接种乳腺癌细胞4T1 1×105 cell/只建立荷瘤小鼠模型。荷瘤小鼠随机分成对照组,DC疫苗组,L-1-MT组,L-1-MT联合DC疫苗组,分别给予生理盐水、DC疫苗、L-1-MT、L-1-MT联合DC疫苗治疗。测量肿瘤大小,描绘肿瘤生长曲线;于接种后第21d处死小鼠,取各组小鼠肿瘤组织,TDLN及脾脏;称取肿瘤重量;Western blot测定TDLN中IDO表达水平,流式细胞仪检测TDLN及脾脏中CD4+CD25+Foxp3+Tregs比例;MTT比色法检测荷瘤小鼠体内T淋巴细胞特异性细胞毒作用。结果:1、4T1冻融抗原致敏的DC(4T1-DC)组表面共刺激分子CD40,CD80,CD86的表达明显高于未致敏DC(DC)组(P0.01);4T1-DC具有更强的刺激T淋巴细胞增殖能力,在刺激细胞与效应细胞比例为1∶10时的刺激指数最高,4T1-DC组、DC组分别为8.45±0.24、6.56±0.29(P0.01);在效应细胞与靶细胞比值为40∶1和20∶1时,4T1-DC组致敏的CTL体外特异性杀瘤效应均高于DC组分别为35.03±1.01、27.02±1.33和31.00±1.06、23.96±1.06(P0.01)。2、接种乳腺癌细胞4T1后的肿瘤生长曲线显示:L-1-MT联合DC疫苗组肿瘤生长速度最慢;第21天时的肿瘤体积和重量均为CON组DC疫苗组L-1-MT组L-1-MT+DC组(P0.05)。L-1-MT+DC联合治疗组与CON组相比,抑瘤率为43%。3、Western blot检测各组TDLN中IDO相对表达量及流式细胞仪测定的TDLN与脾脏中CD4+CD25+Foxp3+Tregs的比例均为DC疫苗组CON组L-1-MT+DC组L-1-MT组(P0.05)。结论:1.应用DC疫苗治疗的荷乳腺癌小鼠TDLN中IDO表达明显增高,且TDLN及脾脏CD4+CD25+Foxp3+Tregs的数量增多,这可能是影响乳腺癌免疫治疗效果的重要因素之一。2.L-1-MT可有效抑制荷乳腺癌小鼠体内IDO蛋白的表达,同时可以下调小鼠TDLN及脾脏内CD4+CD25+Foxp3+Tregs的数量,从而增强DC疫苗抗肿瘤效应有效抑制肿瘤生长。
[Abstract]:Objective: to determine the antitumor effect of dendritic cell (DC) vaccine sensitized by mouse breast cancer cell 4T1 antigen in vitro and in vivo. Objective: to investigate the inhibitory effect of indole 2n 3-dioxygenase (IDO) inhibitor levo-1-methyltryptophan (L-1-MT) combined with DC vaccine on the expression of IDO in tumor drainage lymph nodes (TDLN) of mice bearing breast cancer and the mechanism of enhancing tumor immune response in tumor-bearing mice. Methods the DC derived from bone marrow of BALB/c mice was cultured in vitro and sensitized with 4T1 freeze-thawed antigen to prepare tumor specific DC vaccine. The mature phenotype of DC was detected by flow cytometry and the proliferation ability of T lymphocytes stimulated by DC was detected by mixed lymphocyte reaction (MLR). The specific tumor killing effect of cytotoxic T lymphocyte (CTL) was detected by using T cells sensitized by 4T1 as target cells and DC as effector in vitro. Female BALB/c mice were inoculated with breast cancer cell 4T1 1 脳 10 ~ 5 cell/ under the fourth fat pad to establish tumor bearing mice model. Tumor-bearing mice were randomly divided into two groups: control group, L-1-MT combined with DC vaccine group, and treated with normal saline DC vaccine L-1-MTTL-1-MT combined with DC vaccine respectively. The tumor size was measured, the tumor growth curve was depicted, the mice were killed on the 21st day after inoculation, the tumor tissues and spleen of each group were taken, and the expression of IDO in TDLN was measured by Western blot. The specific cytotoxicity of T lymphocytes in tumor-bearing mice was detected by flow cytometry (FCM), TDLN and CD4 CD25 Foxp3 Tregs ratio in spleen. Results the expression of CD40, CD80, CD80, CD86, a costimulatory molecule in DC (4T1-DC) group, was significantly higher than that in unsensitized DC (DC) group (P0.01). When the ratio of stimulating cells to effector cells was 1:10, the stimulation index was 8.45 卤0.24 卤6.56 卤0.29 (P0.01) in the DC group, and the specific killing effect of CTL in vitro was higher in the ratio of effector cells to target cells at 40:1 and 20:1. The tumor growth curves of DC group were 35.03 卤1.01v 27.02 卤1.33 and 31.00 卤1.060.96 卤1.06 (P0.01) .2.The tumor growth curve after 4T1 inoculation showed that the tumor growth rate was the slowest in the DC vaccine group. On the 21st day, the tumor volume and weight were as follows: CON group, DC vaccine group, L-1-MT DC group (P0.05). L-1-MT DC combined treatment group compared with CON group. The relative expression of IDO in TDLN and the ratio of TDLN to CD4 CD25 Foxp3 Tregs in spleen measured by flow cytometry were all as follows: CON vaccine group, L-1-MT DC group, L-1-MT group (P0.05). Conclusion 1. The expression of IDO in TDLN of breast cancer bearing mice treated with DC vaccine was significantly increased, and the number of TDLN and CD4 CD25 Foxp3 Tregs in spleen was increased. This may be one of the important factors affecting the immunotherapy effect of breast cancer. 2.L-1-MT can effectively inhibit the expression of IDO protein in breast cancer bearing mice, and can down-regulate the number of TDLN and CD4 CD25 Foxp3 Tregs in spleen of mice. In order to enhance the anti-tumor effect of DC vaccine, effectively inhibit tumor growth.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9

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