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目的 探索体外培养日本血吸虫尾蚴细胞的增殖与传代技术。 方法 无菌收集日本血吸虫活尾蚴5 000～10 000条,置于含有10％胎牛血清的RPMI 1640培养基中,用组织刀快速割切尾蚴使成组织碎裂物,加入250 U蟹胶原酶在26℃下孵育30 min,然后离心去酶,加入含有青霉素(100 U/ml)、链霉素(O.1 mg/ml)、两性霉素B(0.25μg/ml)和适量促细胞生长物的RPMI 1640改良培养基中作原代培养,当贴壁细胞增殖长满瓶底时,以1:2的接种率进行传代培养;获取第5代培养细胞作ELISA检测血吸虫病患者血清中抗体。 结果 在原代培养的第3天可见尾蚴组织的周围有呈单个存在或索状排列的发亮细胞,第10天可见单层细胞形成,第14天贴壁细胞长满瓶底并作传代培养;在传代培养中可见细胞呈均匀生长,每7～14 d传代一次;用第5代培养细胞作抗原,检测31例患者的阳性率为90.3％,检测30名正常人血清的假阳性率为6.7％。 结论 日本血吸虫尾蚴细胞体外传代培养至第5代,可用于免疫学研究。
Objective To explore the proliferation and passage of Schistosoma japonicum cercariae cells cultured in vitro. Methods A total of 5 000-10 000 live cercariae of Schistosoma japonicum were collected sterilely in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cercariae were dissected quickly with tissue knife to make tissue fragmentation. 250 U of crab collagenase Incubate at 26 ℃ for 30 min and then remove the enzyme by centrifugation. Add Penicillin (100 U / ml), streptomycin (0.1 mg / ml), amphotericin B When the adherent cells grew well over the bottom of the flask, the cells were subcultured at a 1: 2 inoculation rate. The fifth generation of cultured cells was used for ELISA to detect the antibody in the serum of the schistosomiasis patients . Results On day 3 of primary culture, the cercariae showed the presence of bright cells arranged singly or cord - likely on the 3rd day. Monolayer cells were seen on the 10th day. The adherent cells covered the bottom of the bottle on the 14th day and were subcultured. In subculturing cells showed uniform growth, every 7 ~ 14 d passage once; With the fifth generation of cultured cells as antigen, the positive rate of detection of 31 patients was 90.3%, the detection of 30 normal serum false positive rate was 6.7 %. Conclusion Schistosoma japonicum cercariae cells were subcultured to 5th passage in vitro and could be used in immunological studies.