巨型艾美耳球虫在4种细胞系中培养的比较  

Comparison of Eimeria maxima Culture in Four Cell Lines

作　　者：石梦云 王黎霞[2] 赵小涵 王诗琪 李楠 周爽 张建军[1] 安健[1] SHI Meng-yun;WANG Li-xia;ZHAO Xiao-han;WANG Shi-qi;LI Nan;ZHOU Shuang;ZHANG Jian-jun;AN Jian(College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China;College of Animal Science and Veterinary Medicine,Beijing Vocatioeal College of Agriculture,Beijing 102442,China)

机构地区：[1]北京农学院动物科学技术学院,北京昌平102206 [2]北京农业职业学院畜牧兽医系,北京房山102442

出　　处：《中国兽医杂志》2023年第4期41-46,共6页Chinese Journal of Veterinary Medicine

基　　金：北京市特色高水平骨干专业群项目-动物医学专业群项目-技术平台与社会服务建设项目(PXM2020-157102-000060-15);北京农业职业学院院级科研项目(XY-JT-03);北京农学院教改项目(BUA2021 JG44)。

摘　　要：为了选择适宜巨型艾美耳球虫(Eimeria maxima)培养的细胞系,本试验首先用DEAE-52纤维素纯化E.maxima第3代裂殖子,然后将其与不同传代细胞系叙利亚幼地鼠肾(BHK)细胞、鸡胚成纤维(DF-1)细胞、猪肾(PK-15)细胞、非洲绿猴肾(Vero)细胞分别在37℃和41℃培养,每隔15 min进行苏木精-伊红(H.E.)染色比较裂殖子入侵4种细胞所需时长;将裂殖子与4种细胞培养12 h,利用H.E.染色方法比较裂殖子入侵4种细胞的活力;通过比较裂殖子接种时长和接种剂量的试验筛选裂殖子入侵DF-1细胞的最佳培养条件。结果显示,在41℃培养温度下裂殖子入侵所需时长更短;在37℃和41℃培养温度下,裂殖子对DF-1细胞的入侵率均显著高于其他3种细胞(P<0.05),裂殖子在与4种细胞培养期间均发育为裂殖体并释放出裂殖子,但未发育成卵囊;裂殖子接种DF-1细胞6 h后入侵率极显著高于4 h(P<0.01),接种10 h入侵率最高,但与8 h无显著性差异(P>0.05);当细胞数∶裂殖子数为1∶2.5时入侵率极显著增加(P<0.01);在BHK、DF-1、PK-15、Vero四种细胞系中,DF-1细胞为最适宜进行E.maxima体外培养的细胞,在37℃、5%CO_(2)条件下,用2.5倍细胞数的裂殖子接种细胞,共培养6 h可收集细胞进行相关试验检测。目前鸡球虫体外培养细胞系有限,而且细胞培养条件不统一,所以其入侵和发育存在不确定性,而E.maxima在这方面研究更少,因此,本试验可为探索新的鸡球虫细胞培养体系提供参考。In order to identify the cell line most suitable for Eimeria maxima(E.maxima)culture,this study purified E.maxima's third generation merozoites by DEAE-52 cellulose for subsequent culture in baby hamster Syrian kidney(BHK),chicken UMNSAH/DF-1(DF-1),porcine kidney-15(PK-15),and African green monkey kidney(Vero)cells at 37℃and 41℃,respectively.Hematoxylin eosin(H.E.)staining was performed every 15 min to compare the time required for merozoites to invade the four cells;the merozoite was cultured in the four cells for 12 h,and the invasion of merozoites into four cells was assessed by H.E.staining;by comparing the inoculation time and dose of merozoites,the best culture conditions for the invasion of DF-1 cells by merozoites were selected.The results showed that the time required for merozoite invasion was shorter at 41℃than at 37℃;at the temperature of 37℃and 41℃,the invasion rate of merozoites into DF-1 cells was significantly higher than that of other three cells(P<0.05);during culture in four cells,merozoites all developed into schizonts and released merozoites,but did not develop into oocysts;after inoculation with DF-1 cells for 6 h,the invasion rate of merozoites was significantly higher than that for 4 h(P<0.01),and the invasion rate was the highest at 10 h,but no significant difference with that at 8 h(P>0.05);when the number of cells∶merozoites was 1∶2.5,the invasion rate increased significantly(P<0.01);among BHK,DF-1,PK-15 and Vero cells,DF-1 was the most suitable cell for E.maxima culture in vitro;under the condition of 37℃and 5%CO_(2),the cells were inoculated with 2.5 times the number of merozoites,and the cells were collected for relevant tests after 6 h.Given that a limited number of cells are available for in vitro culture of E.maxima,its cell culture conditions are far from uniform,and its invasion and development remain uncertain,leading to few reported studies on E.maxima,by providing new cell culture system for chicken coccidian,this study opens the way for further investigatio

关 键 词：巨型艾美耳球虫 裂殖子 细胞系选择 H.E.染色 

分 类 号：S855.9[农业科学—临床兽医学]