循环应用低剂量环磷酰胺对黑色素瘤荷瘤小鼠调节性T细胞的影响及抗瘤作用  

Influence of Cyclical Administration with Low-dose Cyclophosphamide on Regulatory T Cells of Melanoma-bearing Mice and Its Antitumor Effect

作　　者：李卫泊[1] 尹昱[2] 蔡建辉[1] 张峻岭[1] 谢绍建[1] 薛平[1] 周保军[1] 严丽[1] 

机构地区：[1]河北医科大学第二医院,石家庄050051 [2]河北省人民医院康复科,石家庄050051

出　　处：《中国生物制品学杂志》2010年第4期403-407,共5页Chinese Journal of Biologicals

基　　金：河北省科技厅博士基金资助项目(04547002D29);河北省医学适用技术跟踪项目(GL200938)

摘　　要：目的观察循环应用低剂量环磷酰胺(CTX)对黑色素瘤荷瘤小鼠调节性T细胞(Treg)的影响及抗瘤作用。方法通过皮下接种黑色素瘤细胞B16制备黑色素瘤荷瘤小鼠模型;对荷瘤小鼠分别经腹腔单次或循环注射CTX,每隔7d给药1次,共3次,应用流式细胞术检测小鼠脾脏中CD4+CD25+Foxp3+Treg的含量;培养小鼠骨髓来源的树突状细胞(DC),将DC与脾T淋巴细胞混合培养,应用ELISA法检测脾T淋巴细胞干扰素γ(IFNγ)的分泌量;同时测量肿瘤的大小,绘制肿瘤生长曲线,并观察各组荷瘤小鼠自身免疫病的发生情况。结果CTX的最佳应用剂量为100mg/kg。随着荷瘤时间的延长,荷瘤小鼠脾脏CD4+CD25+Foxp3+/CD4+的比值呈逐渐升高趋势。单次应用CTX抑制Treg的时间较短,而循环应用CTX能够延长对Treg的抑制,使其维持在相对较低的水平;循环应用CTX显著提高了荷瘤小鼠脾T淋巴细胞IFNγ的分泌水平。单次和循环应用CTX均未能延缓肿瘤的生长,两组小鼠均未见免疫性白斑及明显的化疗毒副反应。结论循环应用CTX能更加有效地调控Treg,从而促进DC对T淋巴细胞的抗原特异性激活,这将会提高DC疫苗的抗瘤效果。Objective To observe the influence of cyclical administration with low-dose cyclophosphamide（CTX）on regulatory T cells（Treg）of melanoma-bearing mice and its antitumor effect. Methods Melanoma-bearing mouse model was established by inoculating s.c. mice with melanoma B16 cells. The model mice were injected i.p. with CTX once or every 7 d for 3 times, and determined for CD4＋CD25＋Foxp3＋ Treg content in spleens by flow cytometry. The onsets of autoimmune diseases of model mice in various groups were observed. Mouse bone marrow-derived dendritic cells （DCs）were cultured, then co-cultured with the splenic T lymphocytes of model mice and determined for the secretion level of IFNγ by ELISA. The size of tumor was measured, based on which a tumor growth curve was plotted. Results The optimal dosage of CTX was 100 mg / kg. The CD4＋CD25＋Foxp3＋ / CD4＋ ratio in spleens of model mice showed an increasing tendency with the increasing time for melanoma-bearing. The inhibitory effect of a single administration with CTX on Treg lasted for a short time. However, the cyclical administration with CTX prolonged the inhibitory effect, maintaining Treg at a relatively low level. Meanwhile, the cyclical administration with CTX increased the secretion level of IFNγ in splenic T lymphocytes of melanoma-bearing mice. Neither a single administration nor cyclical administration with CTX delayed the growth of tumor. No immune white spots or obvious chemotherapy-associated adverse effects were observed in mice treated with CTX by a single administration and cyclical administration. Conclusion The cyclical administration with CTX was more effective for regulating Treg, thus promoted the antigen specific activation of T lymphocytes by DCs and enhanced the anti-tumor effect of DC vaccine.

关 键 词：环磷酰胺 黑色素瘤 调节性T细胞 树突状细胞 

分 类 号：R735.2[医药卫生—肿瘤] R979.1[医药卫生—临床医学]