本文選題：附紅細(xì)胞體　切入點(diǎn)：CD_4　出處：《遼寧醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的 建立附紅細(xì)胞體感染BALB/c小鼠動(dòng)物模型，檢測小鼠外周血及脾臟中CD_4、白細(xì)胞介素-17（IL-17）、白細(xì)胞介素-23（IL-23）、維甲酸相關(guān)孤核受體γ（tRORγt）的動(dòng)態(tài)變化情況，探索CD_4+T細(xì)胞亞群Th17細(xì)胞在本病發(fā)生過程中發(fā)揮的免疫學(xué)作用，為探討附紅細(xì)胞體感染后誘導(dǎo)機(jī)體產(chǎn)生免疫應(yīng)答的機(jī)制提供理論依據(jù)。 方法 無菌采集疑感染附紅細(xì)胞體豬血液30份，經(jīng)鏡檢及PCR鑒定出附紅細(xì)胞體的陽性血液后備用，并同時(shí)采集正常豬血作為陰性對照。建立動(dòng)物模型，將96只BALB/c小鼠隨機(jī)分為四組：A組（陽性組）、B組（陰性組）、C組（純化組）、D組（生理鹽水組），同時(shí)分為四個(gè)時(shí)間點(diǎn)：3d、5d、7d、9d，每組的每個(gè)時(shí)間點(diǎn)6只小鼠。各組小鼠分別經(jīng)腹腔接種0.5ml的豬附紅細(xì)胞體陽性血樣、豬附紅細(xì)胞體陰性血樣、純化附紅細(xì)胞體、生理鹽水。在接種后第3d、5d、7d、9d，采用RT-PCR檢測小鼠脾臟IL-17、IL-23、RORγt mRNA的表達(dá)情況，WesternBlotting檢測小鼠脾臟IL-17的蛋白翻譯情況，熒光雙標(biāo)法檢測小鼠外周血CD_4、IL-17的表達(dá)變化，流式細(xì)胞術(shù)檢測小鼠脾臟CD_4、IL-17的表達(dá)變化。 結(jié)果 1、RT-PCR檢測小鼠脾臟IL-17、IL-23、RORγt mRNA的表達(dá)水平結(jié)果顯示，接種后第3d、5d、7d、9d，A、C組明顯高于B、D組（P＜0.05），A組與C組比較沒有統(tǒng)計(jì)學(xué)差異（P0.05），B組與D組比較沒有統(tǒng)計(jì)學(xué)差異（P0.05）；感染組小鼠IL-17與RORγt mRNA的表達(dá)呈先上升再下降的趨勢，且在接種5d時(shí)其檢測指標(biāo)達(dá)到高峰，但是感染組IL-23mRNA的表達(dá)呈持續(xù)升高的水平。 2、Western Blotting檢測小鼠脾臟IL-17蛋白翻譯水平趨勢結(jié)果同RT-PCR檢測IL-17mRNA轉(zhuǎn)錄水平結(jié)果。 3、熒光雙標(biāo)檢測小鼠外周血CD_4、IL-17表達(dá)水平（Th17陽性表達(dá)=IL-17/CD_4）結(jié)果所示，接種后第3d、5d、7d、9d統(tǒng)計(jì)Th17細(xì)胞陽性表達(dá)率比較，A、C組明顯高于B、D組（P＜0.05），，A組與C組比較沒有統(tǒng)計(jì)學(xué)差異（P0.05），B組與D組比較沒有統(tǒng)計(jì)學(xué)差異（P0.05），且感染組在接種5d時(shí)Th17細(xì)胞陽性表達(dá)達(dá)到高峰。 4、流式細(xì)胞術(shù)檢測小鼠脾臟CD_4、IL-17表達(dá)水平（Th17陽性表達(dá)=IL-17/CD_4）趨勢結(jié)果同熒光雙標(biāo)檢測Th17陽性表達(dá)結(jié)果。 結(jié)論 本實(shí)驗(yàn)通過建立附紅細(xì)胞體感染BALB/c小鼠動(dòng)物模型，證實(shí)附紅細(xì)胞體感染機(jī)體后IL-23、RORγt轉(zhuǎn)錄水平增加，促進(jìn)CD_4+T細(xì)胞開始向Th17細(xì)胞分化并通過分泌炎性細(xì)胞因子IL-17發(fā)揮了積極的抗感染作用，為本病的治療診斷及預(yù)防提供了一定了理論數(shù)據(jù)，開辟了新的思路。
[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, 緯 tROR 緯 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 緯 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 緯 t mRNA in spleen of mice was detected by RT-PCR. The expression of IL-17 and ROR 緯 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 緯 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 緯 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.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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