目的：观察米托蒽醌（mitoxantrone，MIT）对黑素瘤B16细胞钙网蛋白（calreticulin, CRT）表达的影响，探讨高表达CRT的B16细胞膜抗原疫苗的免疫效果及其机制。方法：不同剂量MIT处理B16细胞，免疫荧光法检测B16细胞CRT的表达。以B16细胞建立小鼠荷瘤模型，用不同剂量的MIT治疗荷瘤小鼠，观察MIT对黑素瘤生长及肿瘤组织中CRT表达的影响。制备B16细胞膜蛋白和MIT处理后B16细胞膜蛋白作为疫苗分别免疫小鼠，免疫组化检测小鼠移植瘤组织内免疫细胞的浸润情况，流式细胞术检测荷瘤小鼠脾脏中CD4+、CD8+T细胞比例的变化。结果：MIT可剂量依赖性地上调B16细胞表面CRT的表达，对照组B16细胞表面CRT为（29.40±3.57）%，高剂量MIT处理组为（72.20±2.94）%（P<0.05）；MIT促进移植瘤组织中CRT的表达，对照组为（3.21±1.37），高剂量MIT组为（9.17±106）（P<0.05）。MIT有效抑制小鼠黑素瘤的生长（P<0.05，P<0.01）。与B16细胞膜蛋白疫苗相比，高表达CRT的MITB16细胞膜蛋白疫苗可明显上调小鼠黑素移植瘤组织中的DCs和T细胞的数量，以及脾脏细胞中CD4+、CD8+T细胞的比例（P<0.05）。结论：MIT能够上调CRT在B16细胞表面的表达，高表达CRT的B16细胞膜蛋白疫苗能够提高肿瘤组织中浸润DCs和T细胞的数量，抑制黑素瘤的生长。

Objective:To investigate the effect of mitoxantrone (MIT) on calreticulin (CRT) expression in B16 cells, and to observe the immune effect of B16membrane antigen vaccine highly expressing CRT on B16 tumorbearing mice. Methods:The expression of CRT on membrane of B16 cells was detected by immunofluorescence after treatment with different concentrations of MIT. B16implanted mouse model was established, and the growth of B16implanted tumors and CRT expression in B16implanted tumor tissues were observed after treatment with different concentrations of MIT. Membrane antigen vaccines from both normal B16 cells and MITtreated B16 cells were prepared, and mice were immunized before B16 cell implantation. The infiltration of immune cells into B16 tumor tissues and the ratios of CD4+ and CD8+ T cells in the spleen of B16 tumorbearing mice were examined by immunohistochemistry and flow cytometry, respectively. Results:Flow cytometry results showed that MIT dosedependently increased CRT expression on B16 cell membrane, with CRT expression in control and high dosage MIT groups being (29.40±3.57)% and (72.20±2.94)% (P<0.05), respectively. MIT also increased CRT expression in B16 tumor tissues, with those in the control and high dosage MIT groups being 3.21±1.37 and 9.17±1.06 (P<0.05), respectively. MIT effectively inhibited the growth of B16 tumors (P<0.05). Compared with normal B16 cell membrane antigen vaccine, the vaccine highly expressing CRT increased the numbers of DCs and T cells in B16 tumors tissues and the ratios of CD4+ and CD8+ T cells in the spleen (P<0.05). Conclusion:MIT can increase CRT expression on membrane of B16 cells. B16membrane antigen vaccine highly expressing CRT can enhance the infiltration of DCs and T cells in melanoma, thus improving the immune effect of B16membrane antigen vaccine.

河北省科学技术研究与发展计划资助项目（No.09276418D26）；河北省医学适用技术跟踪项目（No.GL200928）