日本血吸虫尾蚴细胞的传代培养及抗原性检测

Passage Cultivation and Immunological Identification of Schistosoma japonicum Cercaria Cells

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 study the methods of in vitro proliferation and passage cultivation of the cells of Schistosoma japanicum cercariae. Methods Between 5 000 and 10 000 cercariae of Schistosoma japonicum were collected under aseptic condition and placed into RPMI 1640 medium containing 10% fetal bovine serum. The cercariae were disrupted swiftly using a tissue tearor, and the disrupted material was incubated for 30 min in 250 U crab collagenase at 26℃. After centrifugation, the enzyme solution was removed, and modified RPMI 1640 medium was added containing penicillin (100 U/ ml), streptomycin (0.1 mg/ ml), and amphotericin B (0.25 μg/ ml), and certain amount of cell growth enhancing material. When adhesion cells proliferated and grew fully on the bottom, subculture was in progress according to 1:2 split ratio. Cells cultivated by 5 passages were used to detect the specific antibody in sera of patients with chronic schistosomiasis through ELISA. Results On the 3rd day of primary cultivation, bright cells, both individual and clustered, were seen around the disrupted cercariae. A monolayer of cells formed on the 10th day. Adhesion cells grew fully on the bottom and subculture was in progress on the 14th day. Cells were found to grow evenly in passage cultivation, within every 7-14 days another passage could be made. Using cells of the fifth passage cultivation as antigen, the antibody positive rate was 90.3% in patients with chronic schistosomiasis and the false positive rate was 6.7% in healthy controls. Conclusion The in vitro passage cultivation of S. japonicum cercaria cells has been successful to the 5th generation and the cultured material could be used in the immunological research.