日本血吸虫感染鼠肝脏肉芽肿细胞分离条件的优化及分离效果的流式检测  

Optimized method for isolation of hepatic granuloma cells from mice infected with Schistosoma japonicum and verified by Flow Cytometry

作　　者：任丹丹 夏媛媛 王秀男 刘彪 李强[3] 沈际佳[2] 张玉侠[1] REN Dan-dan;XIA Yuan-yuan;WANG Xiu-nan;LIU Biao;LI Qiang;SHEN Ji-jia;ZHANG Yu-xia(Department of Pathology and Pathophysiology,Hefei 230032,China;Department of Microbiology and Parasitology,Hefei 230032,China;College of Life Sciences,Anhui Medical University,Hefei 230032,China)

机构地区：[1]安徽医科大学病理生理学教研室,合肥230032 [2]安徽医科大学病原生物学教研室,合肥230032 [3]安徽医科大学生命科学院,合肥230032

出　　处：《中国人兽共患病学报》2018年第9期778-782,共5页Chinese Journal of Zoonoses

基　　金：安徽省自然基金项目(No.1808085MH269);安徽省教育厅基金项目(No.KJ2018A0168);国家自然科学基金项目(No.81471982)联合资助~~

摘　　要：目的优化日本血吸虫感染小鼠肝脏肉芽肿细胞的分离方法,以获得足量和纯净的肉芽肿活细胞,流式检测确定细胞的分离效果。方法血吸虫尾蚴经皮感染C57BL/6小鼠,于感染后6周、12周剖杀小鼠,取肝脏,采用Ⅳ型胶原酶两步消化法分离肉芽肿细胞,并对胶原酶浓度及消化时间进行探索;进行流式检测,分析粒细胞、淋巴细胞和CD_4^+T的分离效果。结果感染6周小鼠肝脏的Ⅳ型胶原酶最佳浓度为0.25mg/mL,最佳消化时间为30min(第一步)和30min(第二步),流式细胞仪检测该条件可获得较高比例CD_4^+T细胞;而感染12周小鼠的肝脏,Ⅳ型胶原酶最佳浓度为0.3mg/mL,消化时间分别为40、30min,流式检测此条件可获得更高比例的CD_4^+T细胞。结论急慢性肝脏肉芽肿细胞的分离所需Ⅳ型胶原酶的浓度不同;采用本研究优化的胶原酶两步消化法可获得大量、纯净的肉芽肿活细胞,并且细胞表面标志保存完好,为研究日本血吸虫肝脏肉芽肿的细胞机制提供重要材料。We aimed to explore the better method for the separation of sufficient and pure hepatic granulomatous cells from the infected mice by the schistosomes and determined the effects of cell separation by flow cytometry.The mice were infected with cercariae of S.japonicum for 6 weeks or 12 weeks,and then the livers of infected mice were digested inⅣcollagenase solution(exploring the better concentration ofⅣcollagenase solution and digestion duration).Granular cells,lymphocytes and CD 4+T cells were determined by flow cytometry.Our work demonstrateds that the best optimal digestion period was 30 min for both the first and the second digestion for the livers from 6-week infected mice with 0.25 mg/mL collagenase typeⅣto obtain the higher proportion of CD 4+T cells.The livers from 12-week infected mice,however,required a longer digestion of 40 min for the first step and 30 min for the second step with 0.3 mg/mL type IV collagenase.Flow test showed that more CD 4+T cells were obtained.So,the optional concentration and digestion period of collagenase type IV for the isolation hepatice granuloma cells depend on the acute or chronic infection stages.Plenty of pure and viable granulomatous cells with intact cell surface marker were recovered by two stages of collagenase digestion method,providng critical material for studying the cellular mechanism of liver granuloma of S.japonicum.

关 键 词：日本血吸虫 肝脏虫卵肉芽肿 肉芽肿细胞 分离 流式细胞术 

分 类 号：R383.2[医药卫生—医学寄生虫学]