103种化疗药物对犬T细胞功能和毒性的评估

doi:10.16431/j.cnki.1671-7236.2025.09.040

摘要/Abstract

摘要： 【目的】评估化疗药物对犬外周血来源T细胞γ-干扰素(interferon-γ，IFN-γ)分泌、抗肿瘤功能、增殖和杀伤毒性的影响，从而筛选对T细胞毒性和功能影响小的化疗药物，以便单独或联合其他免疫治疗药物用于犬肿瘤疾病的临床治疗。【方法】采集健康犬外周血，通过密度梯度离心法分离得到犬外周血单个核细胞(peripheral blood mononuclear cells，PBMCs)，经体外培养扩增获取犬T细胞；采用流式细胞术分析扩增前后T细胞的比例；将103种肿瘤化疗药物以20 μmol/L的浓度作用于犬T细胞48 h，通过ELISA方法检测经药物作用后犬T细胞IFN-γ的释放水平；结合实验室前期研究数据选出具有抗肿瘤活性且对T细胞IFN-γ分泌影响较小的药物，以20 μmol/L的浓度预处理T细胞12 h后，通过结晶紫染色检测药物对T细胞抗肿瘤功能的影响，并通过细胞毒性试验和LDH试验评估所选化疗药物对T细胞的毒性作用。【结果】本研究成功从犬外周血扩增得到纯度高达99.70%的T细胞。ELISA结果显示，多烯紫杉醇等14种药物(13.59%)能显著促进T细胞分泌IFN-γ(P＜0.05),鸦胆子苦醇等34种药物(33.01%)显著抑制T细胞IFN-γ的释放(P＜0.05)，C8-神经酰胺等55种药物(53.40%)对IFN-γ释放无显著影响(P＞0.05)。结合实验室前期研究数据，选出硫酸长春碱、C8-神经酰胺、2'-O-(2-甲氧基乙基)-胞苷和多烯紫杉醇等10种对T细胞IFN-γ分泌无抑制作用的抗犬乳腺肿瘤化疗药物。进一步研究发现，6-巯嘌呤、三水多烯紫杉醇和2'-O-(2-甲氧基乙基)-胞苷等药物对T细胞抗肿瘤功能无显著影响。其中2'-O-(2-甲氧基乙基)-胞苷对犬T细胞毒性较小，细胞增殖活力维持在85%以上，且未引起显著的细胞死亡。【结论】硫酸长春碱、C8-神经酰胺、2'-O-(2-甲氧基乙基)-胞苷和多烯紫杉醇等化合物对犬T细胞毒性较小，其中2'-O-(2-甲氧基乙基)-胞苷对T细胞毒性小且对其功能未产生显著抑制作用，有望成为犬肿瘤化疗或联合疗法的优选药物。

关键词: 肿瘤免疫; 犬T细胞; 化学治疗; 联合治疗

Abstract: 【Objective】 The purpose of this study was to evaluate the effects of chemotherapeutic drugs on the secretion of interferon-γ (IFN-γ),antitumor function,proliferation and cytotoxicity of T cells derived from peripheral blood of dogs,so as to screen chemotherapeutic drugs with little toxicity and impact on T cell function,in order to use alone or in combination with other immunotherapeutic drugs for the clinical treatment of canine tumor disease. 【Method】 Peripheral blood was collected from healthy dogs,and canine peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation.T cells were cultured and expanded in vitro,and their proportion before and after expansion was verified by flow cytometry.103 chemotherapy drugs were applied to canine T cells at a concentration of 20 μmol/L for 48 h,and the release of IFN-γ was measured by ELISA.Based on the previous experimental data in the laboratory,drugs with anti-tumor activity and a small impact on the secretion of IFN-γ in T cells were selected.After pretreating T cells at a concentration of 20 μmol/L for 12 h,the effect of the drugs on the anti-tumor function of T cells was detected by crystal violet staining,and the toxic effects of the selected chemotherapy drugs on T cells were evaluated in detail through cytotoxicity test and LDH test. 【Result】 Canine peripheral blood T cells were successfully expanded in vitro,reaching 99.70% purity.ELISA results demonstrated that of the 103 drugs tested,14 drugs (13.59%) (including docetaxel) significantly promoted the IFN-γ secretion of T cell (P＜0.05),34 drugs (33.01%) (including brusatol) significantly inhibited the release of IFN-γ (P＜0.05),while 55 drugs (53.40%) (C8-ceramide,etc.) had no statistically significant effect(P＞0.05).Combined with previous experimental data,10 candidate chemotherapy drugs including vinblastine sulfate,C8-ceramide,2'-O-(2-methoxyethyl)-cytidine and docetaxel were identified that preserved IFN-γ production in T cells while maintaining anti-tumor efficacy against canine mammary tumors.Further functional assays confirmed that drugs such as 6-mercaptopurine,docetaxel trihydrate and 2'-O-(2-methoxyethyl)-cytidine did not significantly impair T-cell-mediated antitumor activity.Among them,2'-O-(2-methoxyethyl)-cytidine showed relatively low toxicity to canine T cells,preserving ＞85% proliferation viability while causing no significant cell death. 【Conclusion】 Compounds such as vincristine sulfate,C8-ceramide,2'-O-(2-methoxyethyl)-cytidine and docetaxel had little toxicity on canine T cells.Among them,2'-O-(2-methoxyethyl)-cytidine had low toxicity on T cells and no significant inhibitory effect on their function,and it was expected to become the preferred drug for chemotherapy or combination therapy of canine tumors.

Key words: tumor immunity; canine T lymphocytes; chemotherapy; combination therapy

中图分类号:

秦梦可, 李慧鑫, 李四豪, 张启超, 王荣军, 孟庆大, 谢闪闪. 103种化疗药物对犬T细胞功能和毒性的评估[J]. 中国畜牧兽医, 2025, 52(9): 4453-4463.

QIN Mengke, LI Huixin, LI Sihao, ZHANG Qichao, WANG Rongjun, MENG Qingda, XIE Shanshan. Evaluation of 103 Chemotherapy Drugs on Canine T Cell Function and Toxicity[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(9): 4453-4463.

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