【摘要】 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。更多还原

【Abstract】 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.更多还原