钉螺血淋巴细胞及其功能的研究

Studies on Haemocytes of Oncomelania hupensis

目的研究获取钉螺血淋巴细胞的方法,观察其形态结构,研究其免疫等相关功能。方法参照外周淋巴器官中淋巴细胞的悬浮收集法,获取钉螺血淋巴细胞,经Giemsa染色后显微镜观察其形态。血淋巴细胞经结晶紫染色后分别计数悬浮法、传统压片法及针刺法获取的血淋巴细胞数,并进行方差分析和Dunnett-t检验。取冻融的血淋巴细胞上清进行免疫沉淀、抑菌、吞噬杀菌实验。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析血淋巴细胞蛋白组份相对分子质量(Mr)。结果钉螺血淋巴细胞分为圆形有丝状伪足、嗜酸性圆形无丝状伪足、嗜碱性圆形无丝状伪足和梭形细胞4种形态,平均直径依次约为10．93、6．13、6．08及11．06μm,各约占细胞总数的50％、30％、5％及15％。每只钉螺悬浮法、传统压片法及针刺法获取的血淋巴细胞均数分别为1．50、0．66及0．03万／ml。悬浮法与传统压片法、与针刺法差异有统计学意义(F=281．47,P<0．01)。进一步Dunnett-t检验,悬浮法与压片法、与针刺法的血淋巴细胞总体均数差异有统计学意义(t1=15．67,t2=24．50,两组P<0．01)。血淋巴上清与日本血吸虫虫卵可溶性抗原(SEA)反应出现絮状沉淀．抑菌试验对金黄色葡萄球菌和大肠埃希菌出现明显的抑菌圈,血淋巴细胞对白色念珠菌的吞噬率和杀菌率分别为86％和46％。血淋巴细胞蛋白组份相对分子质量约为Mr112 300、107 100、97 200、73 500、60000及12 000。结论悬浮法可获得大量钉螺血淋巴细胞,其具有沉淀SEA、抑制金黄色葡萄球菌和大肠埃希菌生长。以及吞噬杀灭白色念珠菌等免疫功能。

Objectives To establish method for collecting haemocytes of Oncomelania hupensis and study its morphology and immunological importance. Methods Referring to the method of haemocytes collection from peripheral lymphoid organ, suspension technique was used for collection of haemocytes from snails, which were then Giemsa-stained and observed under microscope. Stained by gentian violet, number of haemocytes was counted and compared with that of conventional squashing method and needling method by ANOVA and Dunnett-t test. Supernatant from freeze thawing haemocytes was applied for the tests of immuno-precipitation, bacteriostasis, and phagocytosis. SDS-PAGE was used to analyze relative molecular mass of protein ingredients. Results Four kinds of haemocytes were found： round cells with filiform filopodia, aeidophilie and basophilie round cells both without filiform filopodia, and spindle cells. The average diameter of the 4 type cells was 10,93, 6,13, 6,08, and 11：06μm, and occupied 50%, 30%, 5%, and 15% respectively, The mean of haemoeytes received from suspension, squashing and needling methods was 15 000, 6 600 and 300/ml respectively, ANOVA analysis showed F=281.47, P〈0,01, and Dunnett-t test revealed t1=15,67, P〈0.01 between suspension and squashing methods, and t2=24.50, P〈0,01 between suspension and needling method. The supernatant of haemoeytes showed precipitation with SEA, bacteriostasis with Staphylococcus aureus and Escherichia coli, and 86% phagocytosis and 46% bactericide to Candida albicans. SDS-PAGE revealed approximate Mr 112 300, 107 100, 972 00, 73 500, 600 00, 12 000 of the protein ingredients of the haemocytes. Conclusions Large amount of haemocytes has been obtained from the snails by suspension method, and the cells show immunological activities.