小鼠肝组织经胶原酶Ⅳ不同时长原位消化后Kupffer细胞的分选效果及其肿瘤细胞吞噬功能观察  

Kupffer cell sorting effect and phagocytosis to tumor cells in mouse liver tissues after in situ digestion with collagenase Ⅳ for different durations

作　　者：李洁 翟克超 刘永达 袁继行 LI Jie;ZHAI Kechao;LIU Yongda;YUAN Jihang(Department of Medical Genetics,College of Basic Medical Sciences,Naval Medical University,Shanghai 200433,China)

机构地区：[1]海军军医大学基础医学院医学遗传学教研室,上海200433

出　　处：《山东医药》2022年第24期35-39,共5页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(92059111,81972738);上海市科技创新行动计划实验动物研究领域项目(22140902100);上海市卫生健康委员会卫生行业临床研究专项课题(20214Y0014)。

摘　　要：目的 观察小鼠肝组织经胶原酶Ⅳ不同时长原位消化后Kupffer细胞的分选效果及其肿瘤细胞吞噬功能。方法 用0.04%胶原酶Ⅳ进行小鼠肝脏原位灌注及肝脏组织消化,根据消化时间长短(5、10、20 min)分为5 min消化组、10 min消化组、20 min消化组。Percoll密度梯度离心后获取肝脏非实质细胞(NPC),通过台盼蓝染色计算细胞活率;免疫细胞磁珠分选(MACS)分选Kupffer细胞后通过台盼蓝染色计数Kupffer细胞总数,除以NPC总数,获得Kupffer细胞MACS分选后占比;光学显微镜观察Kupffer细胞形态学变化;流式细胞术分析Kupffer细胞在MACS分选前占NPC总数的比例,再用Kupffer细胞MACS分选后占比除以分选前占比得到MACS分选得率。体外共培养MACS分选所得Kupffer细胞与肿瘤细胞,流式细胞术检测Kupffer细胞对肿瘤细胞的吞噬功能。结果 胶原酶Ⅳ灌注后的肝组织在0.04%胶原酶Ⅳ中于37℃继续消化5 min时NPC总活率、MACS分选的Kupffer细胞数目及Kupffer细胞占NPC的比例、MACS分选得率和细胞形态分析都显示提取效果更佳;且消化5 min时Kupffer细胞对肿瘤细胞的吞噬活性显著高于消化10 min和20 min消化组。结论 适当的胶原酶Ⅳ消化时间对Kupffer细胞肝脏原位灌注结合MACS提取是有利的,而且也更有助于Kupffer细胞对肿瘤细胞的吞噬作用。Objective To observe the sorting effect and phagocytosis to tumor cells of Kupffer cells after mouse liver in situ digestion with collagenase Ⅳ for different durations.Methods Liver in situ perfusion and liver tissue digestion were performed with 0.04% collagenase Ⅳ.According to the length of digestion time(5,10,20 min),they were divided into 5 min,10 min,and 20 min groups,respectively.After Percoll density gradient centrifugation,the liver non-parenchymal cells(NPCs) were obtained.The cell viability was calculated by trypan blue staining;Kupffer cells were sorted by magnetic activated cell sorting(MACS),and the total number of Kupffer cells was counted by trypan blue,and divided by the total number of NPCs to obtain the proportion of Kupffer cells after MACS.Kupffer cell morphology was analyzed by light microscope;flow cytometry was used to analyze the proportion of Kupffer cells in the total number of NPCs before MACS sorting,and then divided the proportion of Kupffer cells after MACS sorting by the proportion before sorting to obtain the MACS sorting yield;MACS-derived Kupffer cells were co-cultured with tumor cells in vitro,and the phagocytosis of tumor cells by Kupffer cells was detected by flow cytometry.Results After perfusion,when the liver tissue was digested in 0.04% type Ⅳ collagenase at 37 ℃ for 5 min,the total NPC viability,the number and proportion of Kupffer cells in NPCs sorted by MACS,the sorting yield of MACS and cell morphology were better;and the phagocytic activity of Kupffer cells in the 5 min digestion group was significantly higher than those in the 10 min and 20 min digestion groups.Conclusion Appropriate collagenase Ⅳ digestion time is beneficial for extraction of Kupffer cells by the method of liver in situ perfusion combined with MACS,and it also helps in the phagocytosis of tumor cells by Kupffer cells.

关 键 词：胶原酶Ⅳ 肝组织消化方法 Kupffer细胞分选方法 Kupffer细胞吞噬功能 

分 类 号：R575[医药卫生—消化系统]