摘要目的:本研究旨在探讨白细胞介素(IL)-2、IL-15、α-干扰素(IFN-α)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)细胞因子组合体外转录mRNA注射小鼠乳腺癌后的治疗效果。方法:体外转录合成细胞因子IL-2、IFN-α、GM-CSF和IL-15 mRNA组合，并分别在体外转染4T1乳腺癌细胞，检测对应蛋白的表达。使用4T1乳腺癌细胞系构建BALB/c小鼠乳腺癌模型，并对其注射4种mRNA组合，通过酶联免疫吸附试验(ELISA)检测注射后第1、4、7、10天小鼠血液中4种细胞因子的水平；测量注射后小鼠肿瘤的大小；采用免疫组织化学方法检测肿瘤中细胞因子的表达。两组间比较采用独立样本 t检验，采用单因素方差分析进行多组间比较。 结果:Western blot实验结果显示，转染组IL-2、IFN-α、GM-CSF和IL-15的表达分别为均显著高于对照组( tIL-2＝4.170， P<0.05； tIFN＝9.648， P<0.01； tGM-CSF＝16.250， P<0.01； tIL-15＝13.550， P<0.01)。与对照组小鼠比较，体外转录mRNA组合注射小鼠乳腺癌模型后可见注射细胞因子后，第7天IL-2实验组和对照组的表达量分别为(361.34±9.16) pg/ml和(259.62±17.28) pg/ml，差异有统计学意义( t＝7.236， P<0.01)；第7天IFN-α实验组表达量[(81.13±21.16) pg/ml]显著高于对照组[(45.91±12.58) pg/ml， t＝6.500， P<0.01]；GM-CSF在第4天实验组和对照组的表达量分别为(1 182.83±42.16) pg/ml和(45.91±762.19) pg/ml，差异有统计学意义( t＝7.077， P<0.01)；实验组中以上3种细胞因子浓度与对照组差异持续到注射后第10天，但IL-15未见明显升高趋势( P>0.05)。注射疫苗后，实验组肿瘤体积[(1 125.13±79.16) mm 3]显著小于对照组[(3 428.91±73.58) mm 3]，差异有统计学意义( t＝11.930， P<0.05)。 结论:IL-2、IL-15、IFN-α、GM-CSF细胞因子的mRNA组合注射小鼠乳腺癌后，血液及肿瘤中4种细胞因子的表达均升高，增强并产生持久性肿瘤特异性免疫反应，从而抑制乳腺肿瘤的生长。

abstractsObjective:To investigate the therapeutic effect of the in vitro transcription mRNA combination of interleukin (IL)-2, IL-15, interferon-α (IFN-α) and granulocyte macrophage-colony stimulating factor (GM-CSF) in breast cancer. Methods:The mRNA combination of cytokines IL-2, IFN-α, GM-CSF and IL-15 was synthesized in vitro, and the expression of corresponding proteins was detected in the 4T1 breast cancer cells transfected with the mRNA combination. The BALB/c breast cancer model was constructed using the 4T1 cells and injected with four mRNA combinations. The levels of cytokines in the blood of mice were detected by enzyme linked immunosorbent assay (ELISA) on the 1st, 4th, 7th, and 10th day after injection. The tumor size was measured, and the expression of cytokines in breast tumor was detected by immunohistochemistry. T-test was used for comparison between the two groups, and one-way ANOVA was used for comparison among multiple groups. Results:Western blotting showed that the expression levels of IL-2, IFN-α, GM-CSF and IL-15 in transfection group were significantly higher than those in control group ( tIL-2=4.170, P<0.05; tIFN=9.648, P<0.01; tGM-CSF=16.250, P<0.01; tIL-15=13.550, P<0.01). Compared with the control group, the expression level of IL-2 was (361.34±9.16) pg/ml in the experimental group and (259.62±17.28) pg/ml in the control group on the 7th day after the injection of cytokines, and the difference was statistically significant ( t=7.236, P<0.01). On day 7, the expression level of IFN-α in the experimental group was (81.13±21.16) pg/ml, significantly higher than that in the control group [(45.91±12.58) pg/ml, t=6.500, P<0.01]. GM-CSF showed significant difference on the 4th day ( t=7.077, P<0.01), the expression level in the experimental group and the control group was (1 182.83±42.16) pg/ml and (45.91±762.19) pg/ml, respectively. Moreover, the differences in the concentrations of the above three cytokines between the experimental group and the control group lasted until the 10th day after injection. However, there was no significant increase trend of IL-15 ( P>0.05). After injection of the vaccine, the tumor volume of the experimental group [(1 125.13±79.16) mm 3] was significantly smaller than that of the control group [(3 428.91±73.58) mm 3], and the difference was statistically significant ( t=11.930, P<0.05). Conclusion:After the injection of mRNAs combination of IL-2, IL-15, IFN-α and GM-CSF to breast cancer, the expression of the four cytokines in blood and tumor were increased, which enhanced and produced persistent tumor specificity immune response, thereby inhibiting the growth of breast cancer.