人芽囊原虫体外培养及感染小鼠调节性T细胞和细胞因子的动态变化的研究

发布时间：2018-01-02 23:04

本文关键词：人芽囊原虫体外培养及感染小鼠调节性T细胞和细胞因子的动态变化的研究　出处：《广西医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:观察在IMDM单相培养基中人芽囊原虫的体外培养情况,建立人芽囊原虫感染小鼠动物模型;观察小鼠感染人芽囊原虫后不同时间点的外周血中白细胞介素4(IL-4)、白细胞介素10(IL-10)、干扰素γ(IFN-γ)表达水平以及小鼠脾脏CD4~+CD25~+Foxp3~+T细胞数量的动态变化规律,初步探讨人芽囊原虫感染的免疫应答特点。方法:从感染者粪便中分离人芽囊原虫,采用IMDM单相培养基对B.h进行体外连续培养,比较不同pH值、血清浓度及接种量等条件下虫体生长繁殖情况及影响因素;IMDM培养基培养人芽囊原虫,实验组经口感染BABL/c小鼠,4×107个/鼠,对照组经口灌食等量培养液,常规饲养,实验组于感染后第2d,4d,6d,2w,3w,4w分批取外周血和脾脏,酶联免疫吸附法(ELISA)检测不同时间点各组小鼠外周血中IL-4、IL-10、IFN-γ的表达水平;同时取脾脏制备单细胞悬液,用FITC-CD4和APC-25单抗进行细胞表面染色,洗涤后,经打孔液和固定液处理,再用PE-FJK-Foxp3抗体进行核内染色,采用流式细胞仪检测CD4~+CD25~+Foxp3~+T细胞数量的变化。结果: B.h在IMDM培养基中生长繁殖良好,其最适培养条件为:pH值在7.0～8.0,新生牛血清含量大于10%,接种量不小于1×105个原虫/管;青、链霉素1万单位/ml,于37℃条件下厌氧培养,每3～6天转种一次,可以达到长期培养。感染人芽囊原虫的小鼠脾脏CD4~+T细胞数量略有增高但无统计学意义,而感染人芽囊原虫的小鼠脾脏CD4~+CD25~+Foxp3~+T细胞数量比例先一过性下降之后明显升高,在第4w又明显降低,与对照组小鼠脾脏CD4+CD25+Foxp3+T细胞相比,在第2d,4d,2w,3w,4w差异有统计学意义。 IL-4,IL-10水平在感染后1周内先缓慢降低然后再升高,于第2周升至峰值之后再缓慢下降,实验组与对照组比较在第2周差异有统计学意义;IFN-γ水平1周内急速上升又在第2周迅速下降,后缓慢上升,在感染后第6天及第2周的实验组与对照组比较差异有统计学意义。结论:IMDM单相培养基适合B.h的生长繁殖,可以用于诊断及体外长期培养,同时为人芽囊原虫的生活史、致病、感染免疫等后续研究奠定基础;感染人芽囊原虫后免疫应答十分复杂,而CD4~+CD25~+Foxp3~+T细胞是其中的重要组成部分,可能参与了早期的免疫抑制;小鼠感染人芽囊原虫后第1周主要发生Th1细胞免疫应答,之后向Th2细胞免疫应答偏移。
[Abstract]:Objective: To observe the medium of Blastocystis hominis in IMDM phase in vitro, establishment of Blastocystis hominis infection in mice animal model; observation of peripheral blood of mice infected with Blastocystis hominis at different time points after interleukin 4 (IL-4), interleukin 10 (IL-10), interferon gamma (IFN-) the expression level and the number of mouse spleen CD4~+CD25~+Foxp3~+T cells changes, to explore the immune response characteristics of Blastocystis hominis infection.
Methods: the separation of Blastocystis from infected feces, using IMDM single phase medium on B.h cultured in vitro, comparison of different pH values, factors of body growth and effects of serum concentration and inoculation conditions of insects; IMDM culture medium of Blastocystis hominis, experimental group orally infected BABL/c mice. 4 x 107 / rats, control group oral feeding with culture medium, conventional breeding, in the experimental group after infection of 2D, 4D, 6D, 2W, 3W, 4W in peripheral blood and spleen, enzyme-linked immunosorbent assay (ELISA) detection of IL-4, different time points were detected in peripheral blood of mice in IL-10, the expression level of IFN- gamma; at the same time from the spleens of single cell suspension was prepared with FITC-CD4 and APC-25 monoclonal antibody cell surface staining, after washing, the drilling fluid and the fixed solution, then PE-FJK-Foxp3 antibody nuclear staining, the changes of CD4~ +CD25~+Foxp3~+T cells by flow cytometry.
Results: B.h in medium growth well IMDM culture, the optimum culture conditions were: pH value 7 ~ 8, newborn bovine serum content of more than 10%, inoculation amount not less than 1 x 105 protozoa / tube; green, streptomycin 10 thousand units of /ml, on the condition of 37 DEG C under anaerobic incubation, every 3 ~ 6 for a day, we can achieve long-term training.
The infection of Blastocystis hominis in the number of mouse spleen CD4~+T cells increased slightly but was not statistically significant, and the infection of Blastocystis hominis number of mouse spleen CD4~+CD25~+Foxp3~+T cells ratio first once decreased after increased in 4W and decreased significantly compared with the control group of mice spleen CD4+CD25+Foxp3+T cells in 2D, 4D, 2W. 3W, 4W difference was statistically significant.
IL-4, IL-10 levels in 1 weeks after infection to chat slowly decreases and then increases, after the second week rose to the peak and then decreased slowly, between the experimental group and control group was statistically significant difference in second weeks; IFN- levels within 1 weeks of the rapid rise and rapid decline in second weeks, then increased slowly after infection sixth days and 2 weeks of the experiment group and the control group the difference was statistically significant.
Conclusion: for the growth and reproduction of B.h IMDM single culture, can be used for diagnosis and long-term culture in vitro, and life history of blastocystishominis disease, lay the foundation for future research of infection and immunity; infection of Blastocystis hominis after the immune response is very complex, and the CD4~+CD25~+ Foxp3~+T cell is one of the important components, may be involved in the early immune suppression; mice infection mainly occurred Th1 cell immune response after first weeks of Blastocystis hominis, to Th2 cell immune response shift.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R383

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