本文選題：濾泡輔助T細(xì)胞　切入點(diǎn)：變應(yīng)性鼻炎　出處：《華中科技大學(xué)》2013年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：第一部分變應(yīng)性鼻炎小鼠動(dòng)物模型的建立 目的：建立變應(yīng)性鼻炎小鼠動(dòng)物模型,探討變應(yīng)性鼻炎鼻腔粘膜隨鼻內(nèi)激發(fā)時(shí)間不同的組織學(xué)變化。 方法：4～6周齡BALB/c小鼠,用卵清蛋白50μg、氫氧化鋁凝膠5mg加生理鹽水1ml配成混懸液進(jìn)行腹腔注射基礎(chǔ)致敏共7次；5%卵清蛋白生理鹽水滴鼻進(jìn)行鼻腔激發(fā),按組分別連續(xù)激發(fā)10天,20天,30天。對(duì)照組用生理鹽水腹腔注射和滴鼻。觀察小鼠行為學(xué)變化,酶聯(lián)免疫吸附法測(cè)試外周血OVA-sIgE的水平,HE染色觀察鼻粘膜嗜酸性粒細(xì)胞浸潤(rùn)情況,檢測(cè)結(jié)果采用SPSS16.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)處理,組間比較用T檢驗(yàn)。 結(jié)果：造模結(jié)束后小鼠行為學(xué)評(píng)分分別為5.01±1.01、6.43+1.12、8.56+1.64,對(duì)照組0.85+0.23；鼻黏膜內(nèi)EOS計(jì)數(shù)分別為45.91(45.91±5.80) pg/ml、48.81(48.81±3.49) pg/ml、51.38(51.38±3.19) pg/ml對(duì)照組0.69±0.12；血清OVA-sIgE濃度分別45.91(45.91±5.80)pg/ml.48.81(48.81±3.49)pg/ml51.38(51.38±3.19)pg/ml,對(duì)照組為41.71(41.71±2.58)pg/ml。T檢驗(yàn)顯示,實(shí)驗(yàn)組和對(duì)照組之間均存在顯著性差異(P0.05),AR模型組之間差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：變應(yīng)性鼻炎小鼠動(dòng)物模型造模成功,隨著鼻內(nèi)激發(fā)時(shí)間的延長(zhǎng),OVA-sIgE濃度和癥狀有升高趨勢(shì)。 第二部分TFH細(xì)胞在變應(yīng)性鼻炎小鼠模型外周血中的流式細(xì)胞分析 目的：流式細(xì)胞術(shù)分析TFH細(xì)胞在變應(yīng)性鼻炎小鼠模型外周血的變化和意義。 方法：變應(yīng)性鼻炎小鼠模型成功建立后,用紅細(xì)胞裂解液處理得到的外周血單個(gè)核細(xì)胞(PBMC)。CD4-Fitc和CXCR5-PE染色后用流式細(xì)胞儀分析檢查外周血的CD4+CXCR5細(xì)胞的比例,ELISA分析血清總OVA-sIgE水平,用統(tǒng)計(jì)學(xué)軟件SPSS16.0作T檢驗(yàn)和Pearson相關(guān)性分析。 結(jié)果：卵清蛋白激發(fā)AR模型組外周血中TFH細(xì)胞占外周血單個(gè)核細(xì)胞的百分比分別為7.94±2.66%、8.65±3.49%、9.77±3.24%,對(duì)照組為4.97±0.20%,經(jīng)比較差異有顯著性,前者明顯高于后者(T-test,P0.05)；AR模型組之間的百分比隨著免疫激發(fā)時(shí)間延長(zhǎng)的而增加,經(jīng)比較差異有顯著性(T-test, P0.05)。Pearson相關(guān)性分析顯示TFH細(xì)胞的表達(dá)水平與總OVA-sIgE水平呈正相關(guān)(r=0.87,P=0.021)。 結(jié)論：TFH細(xì)胞在變應(yīng)性鼻炎發(fā)病中有重要的作用,可能與IgE水平升高有關(guān)。 第三部分IL-21在變應(yīng)性鼻炎小鼠模型中的表達(dá)及與TFH之間的關(guān)系 目的：探討IL-21分子在變應(yīng)性鼻炎小鼠模型中的表達(dá)及與OVA-sIgE、TFH細(xì)胞的關(guān)系。方法：變應(yīng)性鼻炎BALB/c小鼠造模成功后,ELISA法測(cè)試外周血IL-21、OVA-sIgE的量,流式細(xì)胞術(shù)分析TFH細(xì)胞在外周血單個(gè)核細(xì)胞中的含量，SPSS16.0中Pearson相關(guān)性分析IL-21分子與各組數(shù)據(jù)之間的聯(lián)系,均數(shù)間的比較用T檢驗(yàn)。 結(jié)果：IL-21在對(duì)照組為186.25±11.68pg/ml, AR模型1組為181.13±4.93pg/ml,AR模型2組為168.46±11.04pg/ml, AR模型3組為164.54±10.52pg/ml。各試驗(yàn)組和對(duì)照組小鼠外周血IL-21的表達(dá)經(jīng)比較差異有顯著性意義(T-test,P0.05),試驗(yàn)組IL-21的表達(dá)水平要比正常組低,而且隨著鼻內(nèi)激發(fā)時(shí)間的延長(zhǎng),IL-21的表達(dá)水平逐漸下降；Pearson相關(guān)分析顯示外周血IL-21的表達(dá)水平與CD4+CXCR5+T細(xì)胞比例呈負(fù)相關(guān)(r=-0.80,P=0.048),與血清總OVA-sIgE水平呈負(fù)相關(guān)關(guān)系(r=-0.86,P=0.031)。 結(jié)論：IL-21與TFH細(xì)胞及OVA-sIgE的含量呈負(fù)相關(guān),臨床上可能對(duì)變應(yīng)性鼻炎有一定的治療作用。 第四部分TFH相關(guān)因子在變應(yīng)性鼻炎小鼠模型鼻黏膜中的表達(dá) 目的：探討TFH相關(guān)因子CXCR5及Bcl-6在變應(yīng)性鼻炎小鼠模型鼻粘膜中的表達(dá)及可能意義。 方法：小鼠變應(yīng)性鼻炎模型造模成功后,用免疫組織化學(xué)方法分析CXCR5在小鼠鼻黏膜中的表達(dá)情況。并用Westernblot和PCR的方法對(duì)TFH關(guān)鍵轉(zhuǎn)錄因子Bcl-6進(jìn)行分析,數(shù)據(jù)用醫(yī)學(xué)統(tǒng)計(jì)學(xué)軟件SPSS16.0處理及比較。 結(jié)果：Western blot測(cè)試顯示TFH關(guān)鍵轉(zhuǎn)錄因子的表達(dá)在實(shí)驗(yàn)組鼻黏膜明顯升高,其相對(duì)密度值分別為0.67、0.84、0.98,而對(duì)照組鼻黏膜為0.53。鼻黏膜適時(shí)熒光定量PCR檢測(cè)顯示Bcl-6mRNA的表達(dá)也明顯增高,其擴(kuò)增倍數(shù)分別為1.63、1.77、1.85、1.92,而對(duì)照組鼻黏膜為0.90(T-test, P0.05)。鼻黏膜CXCR5免疫組織化學(xué)分析的結(jié)果顯示實(shí)驗(yàn)組鼻黏膜CXCR5+細(xì)胞數(shù)比對(duì)照組鼻黏膜中CXCR5+細(xì)胞數(shù)明顯增多(T-test, P0.05),廣泛分布在粘膜及粘膜下層中。且隨著鼻內(nèi)激發(fā)時(shí)間的延長(zhǎng)表達(dá)增多。 結(jié)論：變應(yīng)性鼻炎小鼠鼻粘膜中CXCR5及Bcl-6表達(dá)明顯增強(qiáng),可能參與了變應(yīng)性鼻炎的發(fā)病過(guò)程。
[Abstract]:The first part of the animal model of allergic rhinitis in mice
Objective: to establish an animal model of allergic rhinitis in mice and to explore the histological changes of nasal mucosa with different excitation time in the nasal cavity.
Methods: 4~6 week old BALB/c mice were 50 g, Aluminium Hydroxide Gel 5mg plus saline 1ml with mixed intraperitoneal injection of a total of 7 times of sensitized suspension; 5% ova saline nasal nasal stimulation, were excited for 10 days, 20 days, 30 days. The control group with normal saline intraperitoneal injection and intranasal. Observed behavioral changes in mice, serum OVA-sIgE ELISA test level, observe the nasal mucosa infiltration of eosinophils in HE staining, the detection results using SPSS16.0 statistical software for data processing, group compared with the T test.
Results: after the modeling of mouse behavior scores were 5.01 + 1.01,6.43+1.12,8.56+1.64, the control group 0.85+0.23; nasal mucosa EOS counts were 45.91 (45.91 + 5.80) pg/ml, 48.81 (48.81 + 3.49) pg/ml, 51.38 (51.38 + 3.19) pg/ml control group 0.69 + 0.12; serum OVA-sIgE concentrations were 45.91 (45.91 + 5.80) pg/ml.48.81 (48.81 + 3.49) pg/ml51.38 (51.38 + 3.19) pg/ml, the control group was 41.71 (41.71 + 2.58) pg/ml.T test showed that there were significant differences between the experimental group and the control group (P0.05), there were statistically significant differences between the AR model group (P0.05).
Conclusion: the animal model of allergic rhinitis in mice is successful. With the prolongation of the time of nasal stimulation, the concentration and symptoms of OVA-sIgE are increasing.
Flow cytometric analysis of the second part of TFH cells in the peripheral blood of Allergic Rhinitis Mice
Objective: to analyze the changes and significance of TFH cells in the peripheral blood of allergic rhinitis mice model by flow cytometry.
Methods: we successfully established the mouse model of allergic rhinitis, with red blood cell lysate from peripheral blood mononuclear cells (PBMC) and.CD4-Fitc CXCR5-PE staining by flow cytometry analysis of peripheral blood CD4+CXCR5 cell ratio, ELISA analysis of the total serum OVA-sIgE level, T test and Pearson correlation analysis the SPSS16.0 statistical software.
Results: the ova AR model group of peripheral blood TFH cell percentage of peripheral blood mononuclear cells were 7.94 + 2.66%, 8.65 + 3.49%, 9.77 + 3.24%, 0.20% + 4.97 in the control group, the difference was significant, the former is higher than the latter (T-test, P0.05); the percentage between AR model the group increased with the prolonged immune challenge, by comparison with significant difference (T-test, P0.05).Pearson correlation analysis showed positive expression of TFH cells and total OVA-sIgE level (r=0.87, P=0.021).
Conclusion: TFH cells play an important role in the pathogenesis of allergic rhinitis, and may be related to the increase of IgE level.
The expression of the third part of IL-21 in the mice model of allergic rhinitis and the relationship with TFH
Objective: To investigate the IL-21 molecule in the mouse model of allergic rhinitis and the expression of OVA-sIgE and TFH, the relationship between cells. Methods: BALB/c mice allergic rhinitis after successful modeling, peripheral blood IL-21 ELISA test, OVA-sIgE, analysis of the content of TFH cells in peripheral blood mononuclear cells by flow cytometry and between the IL-21 molecules and the set of data link Pearson correlation analysis between SPSS16.0, were compared with T test.
Results: IL-21 in control group was 186.25 + 11.68pg/ml, 1 AR model group was 181.13 + 4.93pg/ml, 2 AR model group was 168.46 + 11.04pg/ml. The expression of AR in model group is 164.54 + 3 10.52pg/ml. of the test group and the control group of mice peripheral blood IL-21 the difference was significant (T-test, P0.05) expression. The level of IL-21 of the experimental group than the normal group, and with the extension of intranasal irradiation time, the expression level of IL-21 decreased gradually; the Pearson correlation analysis showed that the expression level of IL-21 in peripheral blood was negatively correlated with the percentage of CD4+CXCR5+T cells (r=-0.80, P=0.048), and negatively related to the total serum OVA-sIgE levels (r=-0.86, P=0.031).
Conclusion: IL-21 has a negative correlation with the content of TFH cells and OVA-sIgE, and may have a certain therapeutic effect on allergic rhinitis in clinical.
The expression of TFH related factors in the nasal mucosa of Allergic Rhinitis Mice Model in fourth parts
Objective: To investigate the expression and significance of TFH related factors CXCR5 and Bcl-6 in the nasal mucosa of allergic rhinitis mice.
Methods: the mouse model of allergic rhinitis after the success of modeling, analysis and expression of CXCR5 in mouse nasal mucosa by immunohistochemical method. And the TFH key transcription factor Bcl-6 was analyzed with the method of Westernblot and PCR, using medical statistics software SPSS16.0 and compared with the data.
Results: Western blot test showed that the expression of the key transcription factor TFH increased significantly in the experimental group nasal mucosa, the relative density values were 0.67,0.84,0.98, while the control group was 0.53. in nasal mucosa of nasal mucosa of real-time fluorescent quantitative PCR detection showed that the expression of Bcl-6mRNA was also increased, the amplification ratio was 1.63,1.77,1.85,1.92, while the control group was 0.90 in nasal mucosa (T-test, P0.05). Analysis of the nasal mucosa CXCR5 immunohistochemistry results showed that CXCR5+ cells in nasal mucosa of experimental group significantly increased the number of CXCR5+ cells than the control group in nasal mucosa (T-test, P0.05), the number of widely distributed in the mucosa and submucosa. And with nasal challenge time was increased.
Conclusion: the expression of CXCR5 and Bcl-6 in nasal mucosa of allergic rhinitis mice is obviously enhanced, which may be involved in the pathogenesis of allergic rhinitis.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R765.21

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4 謝欣;變應(yīng)性鼻炎：一年四季打噴嚏[N];大眾衛(wèi)生報(bào);2005年

5 國(guó)家中醫(yī)藥管理局適宜技術(shù)推廣項(xiàng)目;按揉法治變應(yīng)性鼻炎[N];中國(guó)中醫(yī)藥報(bào);2009年

6 健康時(shí)報(bào)記者 許曉華;治過(guò)敏至少要堅(jiān)持兩年[N];健康時(shí)報(bào);2008年

7 一文;急性鼻炎變應(yīng)性鼻炎之區(qū)別[N];醫(yī)藥經(jīng)濟(jì)報(bào);2002年

8 ;益肺通竅法治療變應(yīng)性鼻炎[N];中國(guó)中醫(yī)藥報(bào);2003年

9 ;中醫(yī)藥綜合療法治療變應(yīng)性鼻炎效佳[N];中國(guó)中醫(yī)藥報(bào);2004年

10 文;過(guò)敏性鼻炎不可輕視[N];醫(yī)藥經(jīng)濟(jì)報(bào);2002年

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2 滑[

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