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

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CD4+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0.25 mg/mL,最佳消化时间为30 min(第一步)和30 min(第二步),流式细胞仪检测该条件可获得较高比例CD4+T细胞;而感染12周小鼠的肝脏,Ⅳ型胶原酶最佳浓度为0.3 mg/mL,消化时间分别为40、30 min,流式检测此条件可获得更高比例的CD4+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。

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

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 in Ⅳ collagenase solution (exploring the better concentration of Ⅳ collagenase solution and digestion duration).Granular cells, lymphocytes and CD4+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 type Ⅳ to obtain the higher proportion of CD4+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 CD4+ 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.