二乙基亚硝胺诱发大鼠肝癌过程中脾脏巨噬细胞的变化

Changes in rats splenic macrophage during the process of live cancer induced by diethylnitro- samine

目的观察在二乙基亚硝胺（DEN）诱发大鼠肝癌过程中大鼠脾脏巨噬细胞（Mφ）结构与功能的变化。方法将SD大鼠50只随机分为正常对照组10只和实验组40只。实验组按DEN腹腔注射加慢性间断自由饮用DEN水溶液的方法制备肝癌肺转移模型，分别在造模的第8、13、16周随机处死大鼠，根据病理结果依次归为为肝硬化组（10只）、肝癌未转移组（10只）、肝癌转移组（10只）。应用透射电镜观察各组大鼠脾脏Mφ超微结构。贴壁法分离纯化Mφ，应用Vybant吞噬检测试剂盒、噻唑蓝（MTT）比色法、Rat肿瘤坏死因子（TNF）-αElispot试剂盒、DQ^TM ovalbumin试剂盒检测脾脏巨噬细胞活力和吞噬、代谢、分泌及抗原呈递功能。结果Mφ超微结构：肝硬化组和肝癌未转移组Mφ较正常组Mφ表面突起增多，细胞器增多；肝癌转移组Mφ表面突起减少，细胞器较少。Mφ吞噬指数：与正常对照组（75．6±1．7）比较，肝硬化组（84．7±1．9）升高（P〈0．05）；肝癌未转移组（89．5±3．1）显著升高（P〈0．01）；肝癌转移组（36．0±2．6）显著降低（P〈0．01）。Mφ代谢率：与正常对照组（1．48±0．17）比较，肝硬化组和肝癌未转移组代谢率（1．53±0．15、1．56±0．14）增高（P〈0．05），肝癌转移组（1．12±0．29）代谢率降低（P〈0．05）。TNF—α分泌值与正常对照组（626．6±24．6）比较，肝硬化组和肝癌未转移组（分别为741．0±52．9、1126．2±174．5）升高（P〈0．05），肝癌未转移组增高更为显著（P〈0．01），肝癌转移组（313．8±50．8）显著降低（P〈0．01）。抗原呈递阳性细胞比率（％）：与正常组（16．45±1．86）比较，肝硬化组和肝癌未转移组（分别为24．03±1，87、27，95±2．63）显著升高（P〈0．01）；肝癌转移组（10．46±2．16）显著降低（P〈0．01）。结论在肝硬化及肝癌早期，脾脏Mφ吞噬、代谢、分泌、抗原呈递功能普遍增强；在肝癌晚期，脾脏Mφ吞噬、代谢、分泌、抗原呈递功能普遍减弱。说明脾脏抗肿瘤作用呈双向性和时相性。

Objective Background It generally accepted that spleen played complex role in the tumor immunity,which would change in the different periods of cancer. In this study, we investigate the changes in the functions of splenic macrophage （Mφ） in different stages of liver cancer induced by diethylnitrosamine （DEN） in rats, to support the characteristics of ＂two-way＂ and ＂phase＂ of spleen in antitumor immunity. Methods Establishing the mode of pulmonary metastasis of liver cancer in forty male SD rat by diethylnitrosamine. In the 8th, 13th and 16th week, 10 rats were randomly chosen and sacrificed, and divided into cirrhosis, liver cancer and pulmonary metastasis group depending on the pathology result, respectively. The other 10 rat were taken as control group. The Mφ was isolated by anchoring cultivation. The changes in uhrastructure,phagocytosis, cytokine secretion, antigen processing and presenting, and viability of splenic Mφ were detected by transmission electron microscope, VybrantTM Phagocytosis Assay, DQTM Ovalbumin, and human TNF-α ELISpot kits. Results Under the electron microscope,the Mφ in the control group had some prominences like pseudopodium, and mitochondria, ribosome, rough endoplasmic reticulum, lysosome can be found in the cytoplasm, and RBC phagocytosed. In the liver cirrhosis and liver cancer group, Mφ had more prominences, meanwhile much more mitochondria,ribosome, rough endoplasmic reticulum, lysosome can be found in the cytoplasm, especially the liver cancer group. What is more, we can see the macrophage that is phagocytoseing or had phagocytosed other apoptosis and necrosis cells. In the pulmonary metastasis group, the Mφ was swelling, with few organelle. As compared to the control group, the function of splenic Mφ increased in cirrhosis and cancer group, but decreased in metastasis group （ phagocytosis rate ： 84.7 ± 1.9,89.5 ± 3.1 and 36.0 ± 2.6 vs 75.6 ± 1.7, P 〈 0.05, P 〈 0.01 ; viability ： 1.53 ± 0.15,1.56 ± 0.14, and 1.12 ± 0. 29 vs 1.48 ± 0. 17, P 〈 0.05 ; TNF-α secretion ：741.0 ± 52. 9,1126.2 ± 174.5, and 313.8 ± 50.8 vs 626.6 ± 24.6, P 〈 0.05, P 〈 0.01 ; positive cell rate of antigen processing and presenting：24.03 ±1.87,27.95 ±2.63,and 10.46 ±2.16 vs 16.45 ± 1.86,P〈0.01）. Conclusion In the stage of cirrhosis and early cancer, the immune functions of splenic Mφ were reinforcement. It may promote the non-specificity tumor immunity. On opposite, in the stage of pulmonary metastasis, the immune functions of splenic Mφ were impaired. It may lead to the decrease of tumor immunity.