本文選題：HCT-8細(xì)胞　切入點(diǎn)：培養(yǎng)模型　出處：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：雞球蟲病是由多種艾美耳球蟲寄生于雞的腸上皮細(xì)胞內(nèi)所引起的一種寄生性原蟲病。雞球蟲種類不同,寄生部位和致病性亦有差異,其中柔嫩艾美耳球蟲是致病性最強(qiáng)、危害最為嚴(yán)重的一種。近年來隱孢子蟲、弓形蟲和瘧原蟲在入侵機(jī)制的研究方面發(fā)展迅速,研究發(fā)現(xiàn)蟲體入侵會引起宿主細(xì)胞骨架結(jié)構(gòu)的變化。而雞球蟲也是頂復(fù)門原蟲,其入侵機(jī)制是否相似?為了探討這一問題,本試驗(yàn)以HCT-8傳代細(xì)胞建立體外培養(yǎng)模型,對雞球蟲入侵該細(xì)胞的規(guī)律、球蟲接種劑量、培養(yǎng)基類型、血清濃度、pH等培養(yǎng)條件進(jìn)行優(yōu)化;在此基礎(chǔ)上,探索了宿主細(xì)胞骨架結(jié)構(gòu)改變對柔嫩艾美耳球蟲子孢子入侵的影響。1.柔嫩艾美耳球蟲HCT-8細(xì)胞培養(yǎng)模型的建立。通過在24孔細(xì)胞板上接種一定數(shù)量子孢子后不同時(shí)間取樣,采用HE染色后在顯微鏡下觀察球蟲入侵該細(xì)胞后的發(fā)育情況。結(jié)果顯示球蟲子孢子入侵HCT-8細(xì)胞后可以發(fā)育到第2代裂殖子階段,表明建立的模型可以用于球蟲入侵階段的研究。2.柔嫩艾美耳球蟲對HCT-8細(xì)胞入侵條件的優(yōu)化。以Real-time RT PCR方法評價(jià)子孢子最佳入侵時(shí)間、最適接種劑量、培養(yǎng)基種類、pH值及血清濃度對雞球蟲子孢子入侵率的影響。結(jié)果顯示,雞球蟲在體外培養(yǎng)條件下,最佳入侵時(shí)間為6h,子孢子接種劑量為3×105個(gè)/孔時(shí)入侵達(dá)到飽和,當(dāng)培養(yǎng)基為RPMI 1640且pH值為6.5時(shí)子孢子入侵率最高,血清濃度為1%時(shí)最有利于子孢子入侵。試驗(yàn)優(yōu)化了雞球蟲入侵HCT-8細(xì)胞的條件,為雞球蟲入侵機(jī)制的研究奠定了基礎(chǔ)。3.雞球蟲入侵與宿主細(xì)胞骨架結(jié)構(gòu)的關(guān)系。先將一定數(shù)量的柔嫩艾美耳球蟲子孢子接種到HCT-8細(xì)胞,入侵一定時(shí)間后取樣,對樣品細(xì)胞內(nèi)F-actin(肌動(dòng)蛋白)進(jìn)行亞細(xì)胞定位。用不同濃度的細(xì)胞松弛素D抑制宿主細(xì)胞F-actin聚集,免疫熒光染色同時(shí)以Real-time RT PCR方法評價(jià)子孢子對HCT-8細(xì)胞的入侵率。結(jié)果顯示球蟲入侵宿主細(xì)胞過程中,宿主細(xì)胞F-actin特異性聚集在球蟲子孢子表面。球蟲入侵率隨著該藥物濃度增加而下降,當(dāng)濃度達(dá)到50μg/mL時(shí)入侵率下降到原來的20%左右。免疫熒光染色結(jié)果顯示細(xì)胞內(nèi)F-actin聚集不明顯,子孢子附近的F-actin聚集亦不明顯。本試驗(yàn)初步研究了雞球蟲的入侵機(jī)制,為進(jìn)一步研究雞球蟲入侵宿主細(xì)胞的信號傳導(dǎo)機(jī)制打下了基礎(chǔ)。
[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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S858.31

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本文編號：1643621