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目的观察低剂量循环应用环磷酰胺(Cyclophosphamide,CTX)联合树突状细胞(Dendritic cells,DCs)疫苗对小鼠黑色素瘤的治疗效果,并探讨其机制。方法体外培养小鼠骨髓来源的DCs,制备DCs疫苗。通过皮下接种黑色素瘤B16细胞复制黑色素瘤荷瘤鼠模型,将荷瘤小鼠随机分为5组:循环CTX+DCs疫苗组、单次CTX+DCs疫苗组、单纯DCs疫苗组、单纯循环CTX组和单纯荷瘤组,治疗方式为腹腔注射CTX(100 mg/kg)后4 d,经瘤周皮下注射TL-DCs疫苗。每隔7 d治疗1次,共3次。观察肿瘤生长情况、小鼠存活状况、自身免疫病及化疗毒副反应的发生情况;于末次治疗后第5天处死小鼠,采用ELISA法检测各组小鼠血清中IFNγ水平,免疫组化法检测小鼠淋巴结内CD8阳性细胞的数量。结果循环CTX+DCs疫苗组抑瘤效果最明显(P<0.01),该组小鼠平均存活期明显高于其他各组(P<0.01),血清中IFNγ水平明显高于其他各组(P<0.01),淋巴结内CD8阳性细胞数量和平均光密度值均高于其他各组(P<0.01);应用CTX的各治疗组中均未见免疫性白斑等自身免疫病及明显化疗毒副反应。结论循环应用低剂量CTX可规律调控调节性T细胞Tregs,能显著提高DCs疫苗的疗效。
Objective To observe the therapeutic effect of low dose cyclophosphamide (CTX) combined with dendritic cells (DCs) vaccine on mouse melanoma and to explore its mechanism. Methods Mouse bone marrow-derived DCs were cultured in vitro to prepare DCs vaccine. The tumor-bearing mice were randomly divided into 5 groups: CCX + DCs vaccine group, single CTX + DCs vaccine group, DCs alone vaccine group, purely circulating CTX group And simple tumor-bearing group were treated by intraperitoneal injection of TL-DCs subcutaneously 4 days after intraperitoneal injection of CTX (100 mg / kg). Treatment once every 7 days, a total of 3 times. The growth of mice, the survival status of mice, autoimmune diseases and the occurrence of chemotherapy toxicity were observed. On the 5th day after the last treatment, the mice were sacrificed and the levels of IFNγ were detected by ELISA. Immunohistochemistry The number of CD8 positive cells in the mouse lymph nodes was measured. Results The CTX + DCs vaccine group had the most obvious antitumor effect (P <0.01), the average survival time of the mice in the CTX + DCs vaccine group was significantly higher than that in other groups (P <0.01), and the serum IFNγ levels were significantly higher than those in other groups (P < 0.01). The number and average optical density of CD8 positive cells in lymph nodes were higher than those in other groups (P <0.01). No autoimmune diseases such as autoimmune leukoplakia and obvious chemotherapeutic toxicities were observed in all the groups treated with CTX. Conclusion Cyclic low-dose CTX regulates regulatory T cells Tregs regularly, which can significantly improve the efficacy of DCs vaccine.