pCD86-RAE-1ε转基因小鼠的深入鉴定及细胞免疫功能分析

发布时间：2018-01-17 14:32

本文关键词：pCD86-RAE-1ε转基因小鼠的深入鉴定及细胞免疫功能分析　出处：《扬州大学》2011年硕士论文　论文类型：学位论文

【摘要】：机体的天然免疫和获得性免疫紧密联系,NK细胞与树突状细胞(DC)之间的对话(crosstalk)不仅影响天然免疫功能,对获得性免疫的形成、发展及结果有深远的影响。DC通过直接接触NK细胞及分泌细胞因子激活NK细胞,并通过上调NK细胞的活性而增强其抗肿瘤作用。同时活化的NK细胞可以激活DC,进一步影响T细胞的活化及是否向Th1方向发展,从而最终影响机体的免疫防御和免疫监视功能。在DC与NK细胞之间的相互作用中,DC表面MICA分子(或其他NKG2D配体)与NK细胞表面NKG2D受体之间的相互作用,不仅直接介导两者之间的对话,并在MICA阳性肿瘤患者体内发挥着激活NK细胞,诱导DC吞噬凋亡肿瘤碎片并交叉递呈抗原、启动特异性免疫应答的作用。但若DC表面持续高表达NKG2D配体,对NK细胞具有怎样的影响,目前尚不清楚。为此,我们制备了DC细胞持续表达MICA(小鼠为RAE-1ε)分子的pCD86-RAE-1ε转基因小鼠,并完成基因整合的初步筛选。本研究主要对转基因小鼠进行RAE-1ε蛋白表达和组织分布的鉴定,以及相关细胞免疫功能分析。研究内容主要分为以下两个部分：第一部分：pCD86-RAE-1εe转基因小鼠的蛋白表达鉴定目的：进一步明确10只PCR鉴定阳性小鼠体内RAE-1ε蛋白的表达情况。方法：猝死小鼠,收取小鼠肺脏、肝脏、胸腺、肠等组织,通过Western Blot鉴定小鼠肝脏组织是否表达RAE-1ε,免疫组织化学技术检测各组织内RAE-1ε(?)(?)表达情况,并利用流式细胞仪进一步鉴定脾脏单细胞悬液内DC、B细胞、巨噬细胞的表达情况。并在此基础上,对阳性转基因小鼠进行常规的交配传代及常规PCR鉴定。结果：1)在10只F0代转基因小鼠中,得到抗原递呈细胞表面持续表达RAE-1ε蛋白的3只小鼠；2)3只阳性转基因小鼠传代到F4代,后代总的阳性率为51.69%以上,有望得到pCD86-RAE-1ε纯合子转基因小鼠。结论：通过基因整合和蛋白表达水平的筛选,获得3只亲代小鼠,其交配的子代小鼠可于后续的相关研究。第二部分：pCD86-RAE-1ε转基因小鼠的细胞免疫功能分析目的：观察抗原递呈细胞持续表达RAE-1ε对相关细胞免疫功能的影响。方法：首先观察阳性小鼠的生命体征,并取转基因小鼠脾脏、淋巴结制备成单细胞悬液,流式细胞术分别检测NK细胞和CTL细胞数目占总淋巴细胞的比例,表面受体NKG2D的表达,杀伤靶细胞活性,分泌IFN-γ的能力。同时检测CD4+T细胞的比例及表面NKG2D的表达情况。其次,取转基因小鼠脾脏来源的抗原递呈细胞(高表达RAE-1ε),刺激野生型小鼠NK细胞和CTL细胞,流式细胞术检测细胞表面NKG2D受体表达、细胞毒活性及分泌IFN-γ的能力。最后,通过ELISA法比较转基因小鼠与野生型小鼠血清内sRAE-1ε和RAE-1ε抗体含量的差异。结果：除部分阳性转基因小鼠出现脱毛症状外,未发现其他相关的异常生命体征。NK细胞和CTL细胞频率均未见明显变化,且其细胞表面NKG2D的表达明显上调,细胞的杀伤活性及分泌IFN-γ的能力均发生显著性升高；CD4+T细胞频率未发生改变,但表面NKG2D的表达明显上调。利用高表达RAE-1ε的抗原递呈细胞刺激野生型小鼠淋巴细胞,其CTL细胞表面NKG2D表达、细胞毒活性及分泌IFN-y的能力均未发生明显改变；而NK细胞分泌IFN-γ的能力虽然未发生改变,但其NKG2D表达和细胞毒活性均明显上调。这提示高表达RAE-1ε的抗原递呈细胞短期刺激淋巴细胞后,可直接增强NK细胞的活性,但对CTL细胞影响不显著。最后,转基因小鼠与野生型小鼠血清内sRAE-1ε和RAE-1ε抗体含量均没有明显的差异。结论：抗原递呈细胞持续高表达RAE-1ε可能导致小鼠自发性脱毛,并引起小鼠体内CTL细胞免疫功能增强,但这两种现象是否有必然联系,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 - 纬 were detected by flow cytometry . Results : The expression of NKG2D and the ability of NK cells to secrete IFN - 纬 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 .

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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