冷冻联合GM-CSF治疗对前列腺癌小鼠脾脏树突状细胞免疫功能影响的实验观察

Experimental study on the immune functions of splenic dendritic cells after a combined therapy of cryoablation and granniocyte macrophage-colony stimulating factor for prostate cancer

目的评价冷冻联合粒巨细胞集落刺激因子（GM-CSF）治疗对前列腺癌小鼠脾脏树突状细胞（DC）数量及免疫功能的影响。方法复制前列腺癌皮下移植瘤，将动物模型随机分为4组：对照组（A组）、GM-CSF组（B组）、冷冻治疗组（C组）、冷冻联合GM-CSF组（D组）。治疗前、治疗后7、14、21d取睥脏，行免疫组织化学染色，并分离脾脏单细胞悬液，流式细胞术分析DC细胞活化比例，LDH释放实验检测脾脏CD8^＋CTL肿瘤特异性杀伤活性，并观察肺转移率。结果治疗后7d，A、B、C、D组脾脏DC细胞数量分别为（个／每高倍视野）：26．4±1．1、36．6±2．1、25．8±1．3、58．2±1．9（P〈0．05）。脾脏DC活化比例分别为：13．60％±1．67％、9．50％±0．21％、14．40％±1．14％、32．80％±2．39％（P〈0．05）。脾脏CD8^＋CTL特异杀伤活性分别为：7．76％．±0．11％、8．10％±O．92％、9．38％±0．45％、41．68％±0．82％（P〈0．05）。术后21d肺转移发生率A、B、C、D组分别为5／5、4／5、4／5、1／5。结论冷冻联合GM-CSF治疗前列腺癌有助于增加脾脏DC细胞数量及活化比例，增强脾脏CTL特异性抗肿瘤反应，降低肺转移瘤的发生率。

Objective To assess the changes about numbers and immune functions of splenic DC （dendritic cell ） after a combined therapy of cryoablation and GM-CSF （granulocyte macrophage-colony stimulating factor） for prostate cancer. Methods Murine model of prostate cancer was established. And the tumor-bearing mice were divided into 4 groups： control group （Group A）, GM-CSF treatment （Group B）, cryoablation treatment （ Group C） and a combined therapy of cryoablation and GM-CSF （ Group D）. Spleens were sampled before and 7, 14, 21 days after treatment. Immunohistochemistry of DC was performed. And splenic lymphocytes were isolated and their activated percents analyzed by flow cytometry. The tumor-specific cytolytic activity of cytotoxie T lymphocyte （CTL） was measured by LDH （ lactate dehydrogenase ） assay. And the lung metastasis rates were calculated. Results At Day 7 post-treatment, the number of DC per high-power field was 26.4±1.1, 36.6 ±2.1, 25.8 ±1.3 and 58.2 ±1.9（P〈0.05）; the activated percent ofDC 13.60% ±1.67%, 9.50%±0.21%, 14.40%±1.14% and 32.80% ±2.39% （P〈 0. 05）; cytolytic activity of CTL against prostate cancer cells 7.76%±0. 11%, 8. 10% ±0. 92%, 9. 38% ±0. 45% and 41.68% ±0. 82% in Groups A, B and C respectively （P 〈0. 05）. At Day 21 posttreatment, the rate of lung metastasis was 5/5, 4/5, 4/5 and 1/5 respectively. Conclusion A combined therapy of cryoablatiou and GM-CSF may increase the number and activated percent of DC in spleen, enhance the tumor-specific immune responses and decrease lung metastasis rate.