低剂量环磷酰胺联合CIK细胞治疗小鼠肝癌研究

doi:10.3760/cma.j.issn.1673-422X.2016.11.003

摘要/Abstract

摘要： 目的探讨低剂量环磷酰胺（CTX）对肝癌荷瘤小鼠调节性T细胞（Treg）的影响及联合细胞因子诱导的杀伤细胞（CIK）的抗瘤效果。方法建立小鼠皮下肝癌模型，采用随机数字表法随机分为4组（每组35只）：正常对照组、单独荷瘤组、单次CTX组（共1次，100 mg/kg，腹腔注射）、循环CTX组（6 d 1次，共3次，100 mg/kg，腹腔注射），流式细胞术检测小鼠脾脏Treg/CD4+细胞比例变化。取小鼠脾脏单个核细胞培养CIK。观察体内抗肿瘤疗效实验中，将荷瘤小鼠随机分为6个治疗组：荷瘤对照组、单次CTX组、单独CIK组、循环CTX组、单次CTX+CIK组、循环CTX+CIK组，绘制各组肿瘤生长曲线，实验结束剥离瘤块称重，计算抑瘤率。结果单独荷瘤组小鼠脾脏Treg/CD4+细胞比例随荷瘤时间延长而逐渐升高，单次CTX组第10天该比例为(8.95±1.90)%，接近单独荷瘤组(9.25±1.74)%，差异无统计学意义(t=0.374，P=0.714)；而循环CTX组在第19天该比例为(8.99±2.11)%，仍低于单独荷瘤组(16.76±2.02)%，差异有统计学意义(t=8.544，P=0.000)。治疗21 d后，荷瘤对照组、单次CTX组、单独CIK组、循环CTX组、单次CTX+CIK组、循环CTX+CIK组的瘤体积和瘤重分别为（3 800.4±607.5）、（3 764.8±537.7）、（3 352.2±485.4）、（2 076.6±620.2）、（1 867.1±533.6）、（970.7±135.3）mm3和（4.01±0.66）、（3.86±0.74）、（3.80±0.42）、（2.08±0.27）、（1.83±0.93）、（0.86±0.25）g；循环CTX+CIK组小鼠瘤体积及瘤重最小，循环CTX和单次CTX+CIK组对肿瘤抑制效应较弱，单独CIK和单次CTX组未能抑制肿瘤生长，6组间比较差异有统计学意义（F=213.750，P=0.000；F=27.142，P=0.000）。结论循环低剂量CTX联合CIK治疗小鼠肝癌疗效显著，可以为肿瘤治疗提供新思路。

关键词: 环磷酰胺, 肝肿瘤, T淋巴细胞, 调节性, 免疫疗法

Abstract: ObjectiveTo explore the influence of low dose cyclophosphamide (CTX) acting on regulatory T cells (Tregs) of hepatocellular carcinomabearing mice, and the antitumor effect of low dose CTX combined with cytokine induced killer cells (CIKs). MethodsModels of tumorbearing mice were established by subcutaneous inoculation with hepatocellular carcinoma cells. The mice were randomly divided into 4 groups （n=35）, there were normal control group, single tumor group, single CTX group (1 time, 100 mg/kg, intraperitoneal) and cyclical CTX group (once every 6 days, 3 times, 100 mg/kg, intraperitoneal). The changes of the Treg/CD4+ in spleens were detected by flow cytometry. We isolated mononulear cells from spleen of mice to culture CIKs. To study the effect of antitumor in vitro, tumorbearing mice were randomly divided into 6 treatment groups, there were single tumor group, single CTX group, single CIK group, cyclical CTX group, single CTX+CIK group and cyclical CTX+CIK group. The growth curves of tumor were drawn. At the end of the experiment, tumor was separated and weighted. The growth inhibition ratio of tumor was calculated. ResultsWith time prolonged after tumor inoculation, the proportion of Treg/CD4+ increased gradually in spleen of mice in single tumor group. On the 10th day, this proportion of single CTX group was (8.95±1.90)% and the single tumor group was (9.25±1.74)%, and there was no difference between two groups (t=0.374, P=0.714). The proportion in the cyclical CTX group (8.99±2.11)% was still lower than that in the single tumor group (16.76±2.02)％ on the 19th day, and the difference was significant (t=8.544, P=0.000). Treatment for 21 days, the volume and weight of the single tumor group, single CTX group, single CIK group, cyclical CTX group, single CTX+CIK group and cyclical CTX+CIK group were (3 800.4±607.5), (3 764.8±537.7), (3 352.2±485.4), (2 076.6±620.2), (1 867.1±533.6), (970.7±135.3)mm3 and (4.01±0.66), (3.86±0.74), (3.80±0.42), (2.08±0.27), (1.83±0.93), (0.86±0.25)g. The tumor volume and weight were minimum in the cyclical CTX+CIK group. The tumor inhibition effects were weaker in the cyclical CTX and single CTX+CIK groups. But tumor in single CIK and CTX groups were unsuppressed. The differences among the 6 groups had statistically significance (F=213.750, P=0.000; F=27.142, P=0.000). ConclusionCyclical administration of lowdose CTX combined with CIKs has an obvious therapeutic effect on hepatocellular carcinoma bearing mice and can provide a new idea for antitumor therapy.

Key words: Cyclophosphamide, Liver neoplasms, T-lymphocytes, regulatory, Immunotherapy

周海静，杨丽平，王黎明，严祥. 低剂量环磷酰胺联合CIK细胞治疗小鼠肝癌研究[J]. 国际肿瘤学杂志, 2016, 43(11): 812-816.

Zhou Haijing, Yang Liping, Wang Liming, Yan Xiang. Anti-hepatocellular carcinoma effect of low dose cyclophosphamide combined with CIK cells in mice[J]. Journal of International Oncology, 2016, 43(11): 812-816.

参考文献

［1］ Chen X, Du Y, Lin X, et al. CD4+CD25+ regulatory T cells in tumor immunity［J］. Int Immunopharmacol, 2016, 34(34): 244-249. DOI: 10.1016/j.intimp.2016.03.009. ［2］Madondo MT, Quinn M, Plebanski M. Low dose cyclophosphamide: mechanisms of T cell modulation［J］. Cancer Treat Rev, 2016, 42(42): 3-9. DOI: 10.1016/j.ctrv.2015.11.005. ［3］Joshi PS, Liu JQ, Wang Y, et al. Cytokineinduced killer T cells kill immature dendritic cells by TCRindependent and perforindependent mechanisms［J］. J Leukoc Biol, 2006, 80(6): 1345-1353. DOI: 10.1189/jlb.0506305. ［4］ Wang Y, Liu T, Tang W, et al. Hepatocellular carcinoma cells induce regulatory T cells and Lead to poor prognosis via production of transforming growth factorβ1［J］. Cell Physiol Biochem, 2016, 38(1): 306-318. DOI: 10.1159/000438631. ［5］ Ke X, Zhang S, Xu J, et al. Nonsmallcell lung cancerinduced immunosuppression by increased human regulatory T cells via Foxp3 promoter demethylation［J］. Cancer Immunol Immunother, 2016, 65(5): 587-599. DOI: 10.1007/s0026201618256. ［6］ Tao Q, Wang H, Zhai Z. Targeting regulatory T cells in cytokineinduced killer cell cultures (Review)［J］. Biomedical Rep, 2014, 2(3): 317-320. DOI: 10.3892/br.2014.234. ［7］ Li CH, Kuo WH, Chang WC, et al. Activation of regulatory T cells instigates functional downregulation of cytotoxic T lymphocytes in human breast cancer［J］. Immunol Res, 2011, 51(1): 71-79. DOI: 10.1007/s12026-011-8242-x. ［8］ Heylmann D, Bauer M, Becker H, et al. Human CD4+CD25+ regulatory T cells are sensitive to low dose cyclophosphamide: implications for the immune response［J］. PLoS One, 2013, 8(12): e83384. DOI: 10.1371/journal.pone.0083384. ［9］ Zhao J, Cao Y, Lei Z, et al. Selective depletion of CD4+CD25+Foxp3+ regulatory T cells by lowdose cyclophosphamide is explained by reduced intracellular ATP levels［J］. Cancer Res, 2010, 70(12): 4850-4858. DOI: 10.1158/0008-5472.CAN-10-0283. ［10］ Huang X, Huang G, Song H, et al. Preconditioning chemotherapy with paclitaxel and cisplatin enhances the antitumor activity of cytokine inducedkiller cells in a murine lung carcinoma model［J］. Int J Cancer, 2011, 129(3): 648-658. DOI: 10.1002/ijc.25702. ［11］ Lutsiak ME, Semnani RT, De Pascalis R, et al. Inhibition of CD4(+)25+ T regulatory cell function implicated in enhanced immune response by lowdose cyclophosphamide［J］. Blood, 2005, 105(7): 2862-2868. DOI: 10.1182/blood2004062410. ［12］ Ge Y, Domschke C, Stoiber N, et al. Metronomic cyclophosphamide treatment in metastasized breast cancer patients: immunological effects and clinical outcome［J］. Cancer Immunol Immunother, 2012, 61(3): 353-362. DOI: 10.1007/s0026201111063. ［13］ Wu J, Waxman DJ. Metronomic cyclophosphamide eradicates large implanted GL261 gliomas by activating antitumor Cd8(+) Tcell responses and immune memory［J］. Oncoimmunology, 2015, 4(4): e1005521. DOI: 10.1080/2162402X.2015.1005521. ［14］ Cerullo V, Diaconu I, Kangasniemi L, et al. Immunological effects of lowdose cyclophosphamide in cancer patients treated with oncolytic adenovirus［J］. Mol Ther, 2011, 19(9): 1737-1746. DOI: 10.1038/mt.2011.113. ［15］ Le DT, Jaffee EM. Regulatory Tcell modulation using cyclophosphamide in vaccine approaches: a current perspective［J］. Cancer Res, 2012, 72(14): 3439-3444. DOI: 10.1158/00085472.CAN113912. ［16］ Kochenderfer JN, Wilson WH, Janik JE, et al. Eradication of Blineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19［J］. Blood, 2010, 116(20): 4099-4102. DOI: 10.1182/blood-2010-04-281931. ［17］ Linette GP, Stadtmauer EA, Maus MV, et al. Cardiovascular toxicity and titin crossreactivity of affinityenhanced T cells in myeloma and melanoma［J］. Blood, 2013, 122(6): 863-871. DOI: 10.1182/blood-2013-03-490565.

[1]	陈红健, 张素青. 血清miR-24-3p、H2AFX与肝癌患者临床病理特征及术后复发的关系研究[J]. 国际肿瘤学杂志, 2024, 51(6): 344-349.
[2]	高凡, 王萍, 杜超, 褚衍六. 肠道菌群与结直肠癌非手术治疗的相关研究进展[J]. 国际肿瘤学杂志, 2024, 51(6): 376-381.
[3]	范志鹏, 余静, 胡静, 廖正凯, 徐禹, 欧阳雯, 谢丛华. 炎症标志物的变化趋势对一线接受免疫联合化疗的晚期非小细胞肺癌患者预后的预测价值[J]. 国际肿瘤学杂志, 2024, 51(5): 257-266.
[4]	张文馨, 夏泠, 彭晋, 周福祥. 甲胎蛋白升高型胃肝样腺癌1例并文献复习[J]. 国际肿瘤学杂志, 2024, 51(5): 312-315.
[5]	杨毫, 施贵冬, 张程城, 张跃, 张力文, 付茂勇. 信迪利单抗与替雷利珠单抗在进展期食管鳞状细胞癌新辅助治疗中的疗效及安全性对比[J]. 国际肿瘤学杂志, 2024, 51(4): 210-216.
[6]	萨蔷, 徐航程, 王佳玉. 乳腺癌免疫治疗研究进展[J]. 国际肿瘤学杂志, 2024, 51(4): 227-234.
[7]	张栋岩, 王品, 魏秋亚, 邓成伍, 魏相相, 高远飞, 王琛. 索凡替尼靶向联合卡培他滨和奥沙利铂治疗肝内胆管癌术后患者1例及文献复习[J]. 国际肿瘤学杂志, 2024, 51(4): 249-253.
[8]	孙维蔚, 姚学敏, 王鹏健, 王静, 贾敬好. 基于血液学指标探讨免疫治疗晚期非小细胞肺癌预后因素及列线图构建[J]. 国际肿瘤学杂志, 2024, 51(3): 143-150.
[9]	刘玉兰, 井海燕, 孙静, 宋伟, 沙丹. 胃癌免疫治疗疗效预测及预后标志物的研究进展[J]. 国际肿瘤学杂志, 2024, 51(3): 175-180.
[10]	彭琴, 蔡玉婷, 王伟. KPNA2在肝癌中的研究进展[J]. 国际肿瘤学杂志, 2024, 51(3): 181-185.
[11]	孙国宝, 杨倩, 庄庆春, 高斌斌, 孙晓刚, 宋伟, 沙丹. 结直肠癌肝转移组织病理学生长方式研究进展[J]. 国际肿瘤学杂志, 2024, 51(2): 114-118.
[12]	向玉玲, 谭佳杰, 熊远果, 赵丽蓉, 黎晨, 张洪. 脱水淫羊藿素对肝癌细胞增殖、迁移和凋亡的影响[J]. 国际肿瘤学杂志, 2023, 50(9): 513-519.
[13]	崔腾璐, 吕璐, 孙鹏飞. 放疗联合免疫治疗在头颈部鳞状细胞癌治疗中的应用[J]. 国际肿瘤学杂志, 2023, 50(9): 548-552.
[14]	李开春, 丁昌利, 于文艳. 安罗替尼联合特瑞普利单抗治疗晚期肺肉瘤样癌1例[J]. 国际肿瘤学杂志, 2023, 50(8): 511-512.
[15]	过慈良, 江春平, 吴俊华. 肠道菌群与肿瘤免疫治疗[J]. 国际肿瘤学杂志, 2023, 50(7): 432-436.