摘要
研究E.tenella子孢子的纯化方法、接种剂量以及细胞生长状态对E.tenella在鸡胚盲肠上皮细胞体外培养中的影响,为E.tenella鸡胚盲肠上皮细胞体外培养模型的建立提供依据。以子孢子回收率和细胞感染率为指标,对DE52纤维素层析法、蔗糖密度梯度离心法和双层目筛过滤法纯化子孢子的效果进行比较;测定了子孢子接种剂量和细胞生长状态对虫体感染率的影响,并对各发育阶段的虫体进行观察。结果表明,采用双层目筛过滤法可得到纯净的子孢子,并且回收率和接种细胞后24、48h的虫体感染率均显著（P〈0.05）和极显著（P〈0.01）高于DE52纤维素层析法和蔗糖密度梯度离心法;在48孔细胞培养板中,子孢子的最佳感染剂量为每孔10万个;鸡胚盲肠上皮细胞覆盖率为85%~90%且贴壁细胞融合率在30%以上时接种子孢子,各时间段虫体的入侵率较高,且可以观察到E.tenella主要发育阶段的虫体。本研究获得了能使E.tenella在鸡胚盲肠上皮细胞完成整个内生发育的子孢子纯化方法、接种剂量和细胞生长状态。
The aim of this study was to investigate the purification methods and inoculation doses of E.tenella sporozoite and the effect of CEC growth state on E.tenella in vitro to provide the theoretical evidences for the E.tenella model establishment in chicken embryonic cecum epithelial cells（CEC）in vitro culture.The effects of sporozoite purification were compared by using different treatment methods such as DE52 cellulose chromatography,sucrose gradient centrifugation and double-layer mesh filtration by the indicators of recovery rate of sporozoite and infection rate of CEC.The inoculation dosages of sporozoite,coverage rate and fusion rate of CEC were detected,and E.tenella were observed in various developmental stages.The pured sporozoites were obtained by using the double-layer mesh filtration method.The recovery rate and infection rate on 24,48 h after inoculation was significantly（P 〈0.01）higher than those of purified using DE52 cellulose chromatography and sucrose density gradient centrifugation.The optimal infection dosage of sporozoite was 100 000 per well in 48-well plate;invasion rate was high and the parasites were obviously observed in different developmental stages when the sporozoite inoculation was carried out with85%-90% of the CEC coverage and above 30% of cell fusion rate.The study obtained that E.tenella could complete the entire endogenous development in CEC under the optimal conditions of sporozoite purification method,sporozoite inoculation dosage and the cell growth.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2016年第3期454-458,共5页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(30771619
31272536)
关键词
E.TENELLA
鸡胚
盲肠上皮细胞
体外培养
影响因素
E.tenella
the chicken embryo
cecum epithelial cells（CEC）
in vitro culture
effective factors
