發(fā)布時(shí)間：2018-01-02 23:04

  本文關(guān)鍵詞：人芽囊原蟲(chóng)體外培養(yǎng)及感染小鼠調(diào)節(jié)性T細(xì)胞和細(xì)胞因子的動(dòng)態(tài)變化的研究　出處：《廣西醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人芽囊原蟲(chóng) IMDM培養(yǎng)基 體外培養(yǎng) CD4~+CD25~+Foxp3~+T細(xì)胞 細(xì)胞因子 免疫調(diào)節(jié)

【摘要】：目的:觀察在IMDM單相培養(yǎng)基中人芽囊原蟲(chóng)的體外培養(yǎng)情況,建立人芽囊原蟲(chóng)感染小鼠動(dòng)物模型;觀察小鼠感染人芽囊原蟲(chóng)后不同時(shí)間點(diǎn)的外周血中白細(xì)胞介素4(IL-4)、白細(xì)胞介素10(IL-10)、干擾素γ(IFN-γ)表達(dá)水平以及小鼠脾臟CD4~+CD25~+Foxp3~+T細(xì)胞數(shù)量的動(dòng)態(tài)變化規(guī)律,初步探討人芽囊原蟲(chóng)感染的免疫應(yīng)答特點(diǎn)。 方法:從感染者糞便中分離人芽囊原蟲(chóng),采用IMDM單相培養(yǎng)基對(duì)B.h進(jìn)行體外連續(xù)培養(yǎng),比較不同pH值、血清濃度及接種量等條件下蟲(chóng)體生長(zhǎng)繁殖情況及影響因素;IMDM培養(yǎng)基培養(yǎng)人芽囊原蟲(chóng),實(shí)驗(yàn)組經(jīng)口感染BABL/c小鼠,4×107個(gè)/鼠,對(duì)照組經(jīng)口灌食等量培養(yǎng)液,常規(guī)飼養(yǎng),實(shí)驗(yàn)組于感染后第2d,4d,6d,2w,3w,4w分批取外周血和脾臟,酶聯(lián)免疫吸附法(ELISA)檢測(cè)不同時(shí)間點(diǎn)各組小鼠外周血中IL-4、IL-10、IFN-γ的表達(dá)水平;同時(shí)取脾臟制備單細(xì)胞懸液,用FITC-CD4和APC-25單抗進(jìn)行細(xì)胞表面染色,洗滌后,經(jīng)打孔液和固定液處理,再用PE-FJK-Foxp3抗體進(jìn)行核內(nèi)染色,采用流式細(xì)胞儀檢測(cè)CD4~+CD25~+Foxp3~+T細(xì)胞數(shù)量的變化。 結(jié)果: B.h在IMDM培養(yǎng)基中生長(zhǎng)繁殖良好,其最適培養(yǎng)條件為:pH值在7.0～8.0,新生牛血清含量大于10%,接種量不小于1×105個(gè)原蟲(chóng)/管;青、鏈霉素1萬(wàn)單位/ml,于37℃條件下厭氧培養(yǎng),每3～6天轉(zhuǎn)種一次,可以達(dá)到長(zhǎng)期培養(yǎng)。 感染人芽囊原蟲(chóng)的小鼠脾臟CD4~+T細(xì)胞數(shù)量略有增高但無(wú)統(tǒng)計(jì)學(xué)意義,而感染人芽囊原蟲(chóng)的小鼠脾臟CD4~+CD25~+Foxp3~+T細(xì)胞數(shù)量比例先一過(guò)性下降之后明顯升高,在第4w又明顯降低,與對(duì)照組小鼠脾臟CD4+CD25+Foxp3+T細(xì)胞相比,在第2d,4d,2w,3w,4w差異有統(tǒng)計(jì)學(xué)意義。 IL-4,IL-10水平在感染后1周內(nèi)先緩慢降低然后再升高,于第2周升至峰值之后再緩慢下降,實(shí)驗(yàn)組與對(duì)照組比較在第2周差異有統(tǒng)計(jì)學(xué)意義;IFN-γ水平1周內(nèi)急速上升又在第2周迅速下降,后緩慢上升,在感染后第6天及第2周的實(shí)驗(yàn)組與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義。 結(jié)論:IMDM單相培養(yǎng)基適合B.h的生長(zhǎng)繁殖,可以用于診斷及體外長(zhǎng)期培養(yǎng),同時(shí)為人芽囊原蟲(chóng)的生活史、致病、感染免疫等后續(xù)研究奠定基礎(chǔ);感染人芽囊原蟲(chóng)后免疫應(yīng)答十分復(fù)雜,而CD4~+CD25~+Foxp3~+T細(xì)胞是其中的重要組成部分,可能參與了早期的免疫抑制;小鼠感染人芽囊原蟲(chóng)后第1周主要發(fā)生Th1細(xì)胞免疫應(yīng)答,之后向Th2細(xì)胞免疫應(yīng)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.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R383

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本文編號(hào)：1371297