附红细胞体感染小鼠Th17细胞免疫功能的实验研究

发布时间：2018-03-29 22:31

本文选题：附红细胞体　切入点：CD_4　出处：《辽宁医学院》2012年硕士论文

【摘要】：目的建立附红细胞体感染BALB/c小鼠动物模型，检测小鼠外周血及脾脏中CD_4、白细胞介素-17（IL-17）、白细胞介素-23（IL-23）、维甲酸相关孤核受体γ（tRORγt）的动态变化情况，探索CD_4+T细胞亚群Th17细胞在本病发生过程中发挥的免疫学作用，为探讨附红细胞体感染后诱导机体产生免疫应答的机制提供理论依据。方法无菌采集疑感染附红细胞体猪血液30份，经镜检及PCR鉴定出附红细胞体的阳性血液后备用，并同时采集正常猪血作为阴性对照。建立动物模型，将96只BALB/c小鼠随机分为四组：A组（阳性组）、B组（阴性组）、C组（纯化组）、D组（生理盐水组），同时分为四个时间点：3d、5d、7d、9d，每组的每个时间点6只小鼠。各组小鼠分别经腹腔接种0.5ml的猪附红细胞体阳性血样、猪附红细胞体阴性血样、纯化附红细胞体、生理盐水。在接种后第3d、5d、7d、9d，采用RT-PCR检测小鼠脾脏IL-17、IL-23、RORγt mRNA的表达情况，WesternBlotting检测小鼠脾脏IL-17的蛋白翻译情况，荧光双标法检测小鼠外周血CD_4、IL-17的表达变化，流式细胞术检测小鼠脾脏CD_4、IL-17的表达变化。结果 1、RT-PCR检测小鼠脾脏IL-17、IL-23、RORγt mRNA的表达水平结果显示，接种后第3d、5d、7d、9d，A、C组明显高于B、D组（P＜0.05），A组与C组比较没有统计学差异（P0.05），B组与D组比较没有统计学差异（P0.05）；感染组小鼠IL-17与RORγt mRNA的表达呈先上升再下降的趋势，且在接种5d时其检测指标达到高峰，但是感染组IL-23mRNA的表达呈持续升高的水平。 2、Western Blotting检测小鼠脾脏IL-17蛋白翻译水平趋势结果同RT-PCR检测IL-17mRNA转录水平结果。 3、荧光双标检测小鼠外周血CD_4、IL-17表达水平（Th17阳性表达=IL-17/CD_4）结果所示，接种后第3d、5d、7d、9d统计Th17细胞阳性表达率比较，A、C组明显高于B、D组（P＜0.05），，A组与C组比较没有统计学差异（P0.05），B组与D组比较没有统计学差异（P0.05），且感染组在接种5d时Th17细胞阳性表达达到高峰。 4、流式细胞术检测小鼠脾脏CD_4、IL-17表达水平（Th17阳性表达=IL-17/CD_4）趋势结果同荧光双标检测Th17阳性表达结果。结论本实验通过建立附红细胞体感染BALB/c小鼠动物模型，证实附红细胞体感染机体后IL-23、RORγt转录水平增加，促进CD_4+T细胞开始向Th17细胞分化并通过分泌炎性细胞因子IL-17发挥了积极的抗感染作用，为本病的治疗诊断及预防提供了一定了理论数据，开辟了新的思路。
[Abstract]:Purpose. To establish an animal model of BALB/c infection with Eperythrozoon, and to detect the dynamic changes of CD4, IL-17, IL-23, 纬 tROR 纬 T in peripheral blood and spleen of mice. To explore the immunological role of CD_4 T cell subsets Th17 cells in the pathogenesis of Eperythrozoon infection and to provide theoretical basis for the mechanism of immune response induced by Eperythrozoon infection. Method. Thirty porcine blood samples from pigs suspected to be infected with Eperythrozoon were collected without bacteria. The positive blood of Eperythrozoon was identified by microscopic examination and PCR, and the normal pig blood was collected as negative control at the same time to establish animal model. Ninety-six BALB/c mice were randomly divided into four groups: group A (positive group), group B (negative group, group C) (purified group D (normal saline group), divided into four time points: 3 days, 5 days, 7 days after 9 days, each group of 6 mice at each time point. Porcine Eperythrozoon positive blood samples inoculated with 0.5ml intravenously, Eperythrozoon and normal saline were purified from porcine Eperythrozoon negative blood samples. The expression of IL-17, IL-23, ROR 纬 t mRNA in spleen of mice was detected by RT-PCR on the 3rd day and 7th day after inoculation. Western blotting was used to detect the protein translation of IL-17 in the spleen of mice. The expression of IL-17 in peripheral blood of mice was detected by fluorescence double labeling method, and the expression of IL-17 in spleen of mice was detected by flow cytometry. Results. 1the expression of IL-17, IL-23 and ROR 纬 t mRNA in spleen of mice was detected by RT-PCR. The expression of IL-17 and ROR 纬 t mRNA in infected mice increased first and then decreased after inoculation. There was no statistical difference between group A and group C (P < 0.05) and group C (P < 0.05), and the expression of IL-17 and ROR 纬 t mRNA in infected group showed a tendency of first increasing and then decreasing. After 5 days of inoculation, the detection index reached the peak, but the expression of IL-23mRNA in the infected group was continuously increased. 2the trend of IL-17 protein translation level in mouse spleen was detected by Western Blotting and IL-17mRNA transcription level was detected by RT-PCR. (3) the level of IL-17 expression in peripheral blood of mice was detected by fluorescence double labeling and the expression of IL-17 + IL-17 / CD4 in peripheral blood of mice was confirmed by the results of Th 17 positive expression and IL-17 / CD4. The positive expression rate of Th17 cells in group A was significantly higher than that in group B (P < 0.05) and group C (P < 0.05). There was no statistical difference between group B and group D. there was no statistical difference between group B and group D, and the positive expression of Th17 cells in infected group reached its peak at 5 days after inoculation. (4) the expression level of IL-17 in spleen of mice was detected by flow cytometry. The trend of Th 17 positive expression of IL-17 + IL-17 / CD4 was compared with that of Th17 positive expression detected by fluorescence double labeling. Conclusion. In this experiment, we established the animal model of BALB/c infection with Eperythrozoon, and confirmed that the transcription level of IL-23 ROR 纬 t increased after Eperythrozoon infection. Promoting the differentiation of CD_4 T cells into Th17 cells and playing an active role in anti-infection by secreting inflammatory cytokine IL-17 provides certain theoretical data for the treatment diagnosis and prevention of this disease and opens up a new way of thinking.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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