本文關(guān)鍵詞：pCD86-RAE-1ε轉(zhuǎn)基因小鼠的深入鑒定及細胞免疫功能分析　出處：《揚州大學》2011年碩士論文　論文類型：學位論文

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【摘要】：機體的天然免疫和獲得性免疫緊密聯(lián)系,NK細胞與樹突狀細胞(DC)之間的對話(crosstalk)不僅影響天然免疫功能,對獲得性免疫的形成、發(fā)展及結(jié)果有深遠的影響。DC通過直接接觸NK細胞及分泌細胞因子激活NK細胞,并通過上調(diào)NK細胞的活性而增強其抗腫瘤作用。同時活化的NK細胞可以激活DC,進一步影響T細胞的活化及是否向Th1方向發(fā)展,從而最終影響機體的免疫防御和免疫監(jiān)視功能。 在DC與NK細胞之間的相互作用中,DC表面MICA分子(或其他NKG2D配體)與NK細胞表面NKG2D受體之間的相互作用,不僅直接介導兩者之間的對話,并在MICA陽性腫瘤患者體內(nèi)發(fā)揮著激活NK細胞,誘導DC吞噬凋亡腫瘤碎片并交叉遞呈抗原、啟動特異性免疫應答的作用。但若DC表面持續(xù)高表達NKG2D配體,對NK細胞具有怎樣的影響,目前尚不清楚。 為此,我們制備了DC細胞持續(xù)表達MICA(小鼠為RAE-1ε)分子的pCD86-RAE-1ε轉(zhuǎn)基因小鼠,并完成基因整合的初步篩選。本研究主要對轉(zhuǎn)基因小鼠進行RAE-1ε蛋白表達和組織分布的鑒定,以及相關(guān)細胞免疫功能分析。研究內(nèi)容主要分為以下兩個部分： 第一部分：pCD86-RAE-1εe轉(zhuǎn)基因小鼠的蛋白表達鑒定 目的：進一步明確10只PCR鑒定陽性小鼠體內(nèi)RAE-1ε蛋白的表達情況。 方法：猝死小鼠,收取小鼠肺臟、肝臟、胸腺、腸等組織,通過Western Blot鑒定小鼠肝臟組織是否表達RAE-1ε,免疫組織化學技術(shù)檢測各組織內(nèi)RAE-1ε(?)(?)表達情況,并利用流式細胞儀進一步鑒定脾臟單細胞懸液內(nèi)DC、B細胞、巨噬細胞的表達情況。并在此基礎(chǔ)上,對陽性轉(zhuǎn)基因小鼠進行常規(guī)的交配傳代及常規(guī)PCR鑒定。 結(jié)果：1)在10只F0代轉(zhuǎn)基因小鼠中,得到抗原遞呈細胞表面持續(xù)表達RAE-1ε蛋白的3只小鼠；2)3只陽性轉(zhuǎn)基因小鼠傳代到F4代,后代總的陽性率為51.69%以上,有望得到pCD86-RAE-1ε純合子轉(zhuǎn)基因小鼠。 結(jié)論：通過基因整合和蛋白表達水平的篩選,獲得3只親代小鼠,其交配的子代小鼠可于后續(xù)的相關(guān)研究。 第二部分：pCD86-RAE-1ε轉(zhuǎn)基因小鼠的細胞免疫功能分析 目的：觀察抗原遞呈細胞持續(xù)表達RAE-1ε對相關(guān)細胞免疫功能的影響。 方法：首先觀察陽性小鼠的生命體征,并取轉(zhuǎn)基因小鼠脾臟、淋巴結(jié)制備成單細胞懸液,流式細胞術(shù)分別檢測NK細胞和CTL細胞數(shù)目占總淋巴細胞的比例,表面受體NKG2D的表達,殺傷靶細胞活性,分泌IFN-γ的能力。同時檢測CD4+T細胞的比例及表面NKG2D的表達情況。其次,取轉(zhuǎn)基因小鼠脾臟來源的抗原遞呈細胞(高表達RAE-1ε),刺激野生型小鼠NK細胞和CTL細胞,流式細胞術(shù)檢測細胞表面NKG2D受體表達、細胞毒活性及分泌IFN-γ的能力。最后,通過ELISA法比較轉(zhuǎn)基因小鼠與野生型小鼠血清內(nèi)sRAE-1ε和RAE-1ε抗體含量的差異。 結(jié)果：除部分陽性轉(zhuǎn)基因小鼠出現(xiàn)脫毛癥狀外,未發(fā)現(xiàn)其他相關(guān)的異常生命體征。NK細胞和CTL細胞頻率均未見明顯變化,且其細胞表面NKG2D的表達明顯上調(diào),細胞的殺傷活性及分泌IFN-γ的能力均發(fā)生顯著性升高；CD4+T細胞頻率未發(fā)生改變,但表面NKG2D的表達明顯上調(diào)。利用高表達RAE-1ε的抗原遞呈細胞刺激野生型小鼠淋巴細胞,其CTL細胞表面NKG2D表達、細胞毒活性及分泌IFN-y的能力均未發(fā)生明顯改變；而NK細胞分泌IFN-γ的能力雖然未發(fā)生改變,但其NKG2D表達和細胞毒活性均明顯上調(diào)。這提示高表達RAE-1ε的抗原遞呈細胞短期刺激淋巴細胞后,可直接增強NK細胞的活性,但對CTL細胞影響不顯著。最后,轉(zhuǎn)基因小鼠與野生型小鼠血清內(nèi)sRAE-1ε和RAE-1ε抗體含量均沒有明顯的差異。 結(jié)論：抗原遞呈細胞持續(xù)高表達RAE-1ε可能導致小鼠自發(fā)性脫毛,并引起小鼠體內(nèi)CTL細胞免疫功能增強,但這兩種現(xiàn)象是否有必然聯(lián)系,期待深入研究。
[Abstract]:DCs can activate NK cells through direct contact with NK cells and secrete cytokines , and enhance the antitumor effect of NK cells by increasing the activity of NK cells . The activated NK cells can activate DC , further influence the activation of T cells and develop in Th1 direction , which can ultimately affect the immune defense and immune surveillance functions of the organism . In the interaction between DC and NK cells , the interaction between MICA molecule ( or other NKG2D ligand ) and NKG2D receptor on NK cell surface is not only mediated directly , but also plays a role in activating NK cells in MICA positive tumor patients , inducing DC to swallow apoptotic tumor fragments and cross - presenting antigen to initiate specific immune response . In this study , we have prepared the transgenic mice expressing MICA ( mouse - 1 蔚 ) molecules in DC cells , and completed the preliminary screening of gene integration . Part I : Identification of Protein Expression in Transgenic Mice with pCD86 - rae Objective : To further clarify the expression of a 10 - PCR in the identification of the E - 1 蔚 protein in the positive mice . Methods : The mice were killed and the lung , liver , thymus , intestine and other tissues of mice were collected . The expression of the mouse liver tissues was determined by Western Blot . The expression of the total internal DC , B cells and macrophages was detected by flow cytometry . On the basis of this , the normal mating and routine PCR identification were performed on the positive transgenic mice . Results : 1 ) In 10 F0 - generation transgenic mice , 3 mice expressing the surface of the antigen - presenting cells were continuously expressed , 2 ) 3 positive transgenic mice were passaged to F4 generation , the total positive rate of the offspring was 51.69 % , and it was hoped that the transgenic mice with pCD86 - rae . epsilon . homozygotes were obtained . Conclusion : Three parent mice were obtained by screening gene integration and protein expression level , and their mating progeny mice could be used in the subsequent study . Part Two : Analysis of Cellular Immune Function in Transgenic Mice with pCD86 - rae Objective : To observe the effect of antigen - presenting cells on the immune function of related cellular cells . Methods : The vital signs of the positive mice were observed and the spleen and lymph nodes of the transgenic mice were taken as single cell suspension . The expression of NK cells and CTL cells was detected by flow cytometry . The expression of NKG2D was detected by flow cytometry . The expression of NKG2D receptor , cytotoxic activity and IFN - 緯 were detected by flow cytometry . Results : The expression of NKG2D and the ability of NK cells to secrete IFN - 緯 were not changed significantly , but the expression of NKG2D was obviously up - regulated . Conclusion : It is necessary to study the immune function of CTL cells in mice , which may lead to spontaneous hair loss in mice , and to increase the immune function of CTL cells in mice .

【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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本文編號：1436661