宿主细胞F-actin聚集与鸡球虫入侵关系研究

发布时间：2018-03-21 11:20

本文选题：HCT-8细胞　切入点：培养模型　出处：《华南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：鸡球虫病是由多种艾美耳球虫寄生于鸡的肠上皮细胞内所引起的一种寄生性原虫病。鸡球虫种类不同,寄生部位和致病性亦有差异,其中柔嫩艾美耳球虫是致病性最强、危害最为严重的一种。近年来隐孢子虫、弓形虫和疟原虫在入侵机制的研究方面发展迅速,研究发现虫体入侵会引起宿主细胞骨架结构的变化。而鸡球虫也是顶复门原虫,其入侵机制是否相似?为了探讨这一问题,本试验以HCT-8传代细胞建立体外培养模型,对鸡球虫入侵该细胞的规律、球虫接种剂量、培养基类型、血清浓度、pH等培养条件进行优化;在此基础上,探索了宿主细胞骨架结构改变对柔嫩艾美耳球虫子孢子入侵的影响。1.柔嫩艾美耳球虫HCT-8细胞培养模型的建立。通过在24孔细胞板上接种一定数量子孢子后不同时间取样,采用HE染色后在显微镜下观察球虫入侵该细胞后的发育情况。结果显示球虫子孢子入侵HCT-8细胞后可以发育到第2代裂殖子阶段,表明建立的模型可以用于球虫入侵阶段的研究。2.柔嫩艾美耳球虫对HCT-8细胞入侵条件的优化。以Real-time RT PCR方法评价子孢子最佳入侵时间、最适接种剂量、培养基种类、pH值及血清浓度对鸡球虫子孢子入侵率的影响。结果显示,鸡球虫在体外培养条件下,最佳入侵时间为6h,子孢子接种剂量为3×105个/孔时入侵达到饱和,当培养基为RPMI 1640且pH值为6.5时子孢子入侵率最高,血清浓度为1%时最有利于子孢子入侵。试验优化了鸡球虫入侵HCT-8细胞的条件,为鸡球虫入侵机制的研究奠定了基础。3.鸡球虫入侵与宿主细胞骨架结构的关系。先将一定数量的柔嫩艾美耳球虫子孢子接种到HCT-8细胞,入侵一定时间后取样,对样品细胞内F-actin(肌动蛋白)进行亚细胞定位。用不同浓度的细胞松弛素D抑制宿主细胞F-actin聚集,免疫荧光染色同时以Real-time RT PCR方法评价子孢子对HCT-8细胞的入侵率。结果显示球虫入侵宿主细胞过程中,宿主细胞F-actin特异性聚集在球虫子孢子表面。球虫入侵率随着该药物浓度增加而下降,当浓度达到50μg/mL时入侵率下降到原来的20%左右。免疫荧光染色结果显示细胞内F-actin聚集不明显,子孢子附近的F-actin聚集亦不明显。本试验初步研究了鸡球虫的入侵机制,为进一步研究鸡球虫入侵宿主细胞的信号传导机制打下了基础。
[Abstract]:Chicken coccidiosis is a kind of parasitic protozoa caused by many kinds of Eimeria infestation in the intestinal epithelial cells of chicken. The parasitic site and pathogenicity of chicken coccidiosis are different with different species of coccidiosis, among which Eimeria tenella is the most pathogenic. In recent years, Cryptosporidium, Toxoplasma gondii and Plasmodium have developed rapidly in the study of invasion mechanism. It has been found that the invasion of parasites will cause changes in the cytoskeleton structure of the host cells. Is its intrusion mechanism similar? In order to study this problem, the culture model of HCT-8 passage cells was established in vitro to optimize the culture conditions such as the invasion of chicken coccidia, the inoculation dose of coccidiosis, the type of culture medium, the concentration of serum and pH, etc. The effects of host cytoskeleton structure change on spores invasion of Eimeria tenella were investigated. 1. Establishment of HCT-8 cell culture model of Eimeria tenella. Samples were taken at different time after inoculating a certain number of sporozoites on the 24 well cell plate. The development of coccidia after invading the cells was observed by HE staining. The results showed that the spores could develop to the stage of the second generation of merozoites after invading the HCT-8 cells. The results showed that the established model could be used in the study of the invasion stage of coccidiosis. 2. Optimization of the invasion conditions of Eimeria tenella to HCT-8 cells. Real-time RT PCR method was used to evaluate the optimal invasion time and the optimal inoculation dose. The effect of pH value and serum concentration on spores invasion rate of Chicken coccidia was studied. The results showed that the optimal invasion time was 6 h and the inoculation dose was 3 脳 105 / well. When the medium was RPMI 1640 and pH was 6.5, the rate of sporozoite invasion was the highest, and the concentration of serum was 1. The conditions of chicken coccidia invading HCT-8 cells were optimized. The relationship between the invasion of chicken coccidiosis and the cytoskeleton structure of host cells. First, a certain number of Eimeria tenella spores were inoculated into HCT-8 cells. The subcellular localization of F-actin (actin) in the sample cells was studied. Different concentrations of cytochalasin D inhibited the aggregation of F-actin in host cells. Immunofluorescence staining was used to evaluate the invasion rate of HCT-8 cells by Real-time RT PCR. When the concentration of F-actin was 50 渭 g / mL, the invasion rate decreased to about 20%. The results of immunofluorescence staining showed that F-actin accumulation in the cells was not obvious. The accumulation of F-actin in the vicinity of the sporozoites was not obvious. The invasion mechanism of chicken coccidiosis was studied preliminarily, which laid a foundation for further study on the signal transduction mechanism of chicken coccidia invading host cells.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.31

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