裸鼹鼠和小鼠巨噬细胞的比较研究

发布时间：2018-03-21 15:17

  本文选题：裸鼹鼠　切入点：小鼠　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的:裸鼹鼠是一种终年生活在地下、营真社会性生活的啮齿类动物,多分布于非洲肯尼亚、埃塞俄比亚等地区。1967年,裸鼹鼠被国外学者引进实验室,半个世纪以来,国内外学者对其展开了一系列的研究,研究报道相继发表于Nature、Science等期刊。在已有的报道中,国内外学者针对裸鼹鼠开展的研究多集中于其寿命长、耐受低氧、缺少疼痛物质、自发性肿瘤鲜有等方面,但是对其免疫系统的研究报道甚少。免疫系统是生物医学研究的重要指标,不仅对动物生理状态的判断和饲养管理具有指导意义,也是临床医疗和科学研究的重要依据。目前已有研究报告揭示免疫系统与癌症之间的关系:随着机体的衰老,其免疫系统也逐渐恶化,这个过程即为免疫衰老,而免疫衰老与全身性炎症、慢性炎症性疾病以及许多癌症相关。因此,本研究率先针对裸鼹鼠的免疫系统进行初步研究,从免疫学的角度来阐释裸鼹鼠寿命长、肿瘤发生率低等生物学特性的机制,进而为人类老年疾病、炎症性疾病及肿瘤的发病机制提供理论依据,同时也为老年疾病、炎症性疾病及肿瘤等疾病的治疗提供新的思路和想法。研究方法:本研究分别选取机体最大的免疫器官脾脏和机体重要的免疫细胞巨噬细胞进行裸鼹鼠免疫系统的初步研究。首先,分离裸鼹鼠和小鼠脾脏淋巴细胞,采用流式细胞术比较裸鼹鼠和小鼠脾脏中免疫细胞的比例。其次,建立裸鼹鼠骨髓来源巨噬细胞分离培养的方法,采用流式细胞术比较裸鼹鼠和小鼠骨髓来源巨噬细胞的吞噬功能。再次,polyI:C模拟病毒感染骨髓来源巨噬细胞,采用流式细胞术比较刺激前后裸鼹鼠和小鼠骨髓来源巨噬细胞的凋亡情况,采用荧光定量PCR技术比较刺激前后裸鼹鼠和小鼠骨髓来源巨噬细胞TLR3和IFN-β表达量的变化情况,进一步采用蛋白免疫印迹技术比较刺激前后裸鼹鼠和小鼠骨髓来源巨噬细胞TLR3和NF-κB表达量的变化情况,研究裸鼹鼠巨噬细胞在抵抗病毒入侵中可能存在的机制。最后,LPS模拟革兰氏阴性细菌感染骨髓来源巨噬细胞,采用流式细胞术比较刺激前后裸鼹鼠和小鼠骨髓来源巨噬细胞的凋亡情况,采用荧光定量PCR技术比较刺激前后裸鼹鼠和小鼠骨髓来源巨噬细胞TLR4和TNF-α表达量的变化情况,进一步采用蛋白免疫印迹技术比较刺激前后裸鼹鼠和小鼠骨髓来源巨噬细胞TLR4和NF-κB表达量的变化情况,研究裸鼹鼠巨噬细胞在抵抗细菌感染中可能存在的机制。结果:脾脏淋巴细胞中免疫细胞比例比较分离裸鼹鼠和小鼠脾脏淋巴细胞,采用流式细胞术检测各类免疫细胞的比例,结果如下:小鼠脾脏巨噬细胞的比例为15.54±2.12%,b细胞的比例为43.21±9.34%,t细胞的比例为37.54±4.50%,nk细胞的比例为6.06±0.23%;裸鼹鼠脾脏巨噬细胞的比例为34.96±5.85%,b细胞的比例为2.34±0.67%,t细胞的比例为32.14±4.66%,nk细胞的比例为3.09±0.16%。和小鼠相比较,裸鼹鼠脾脏淋巴细胞中巨噬细胞的比例较高,而b细胞的比例较低,t细胞和nk细胞的比例基本无差异。建立骨髓来源巨噬细胞分离培养的方法及吞噬功能比较参照经典的小鼠骨髓来源巨噬细胞分离培养的方法,首先分离裸鼹鼠骨髓干细胞,然后利用粒细胞-巨噬细胞集落刺激因子诱导造血干细胞转化为巨噬细胞,并采用流式细胞术进行纯度的鉴定,检测细胞表面蛋白cd11b比例高达90%,鉴定分离培养的细胞为骨髓来源巨噬细胞。分别采用吞噬红细胞法和流式细胞术检测裸鼹鼠和小鼠骨髓来源巨噬细胞的吞噬率,裸鼹鼠骨髓来源巨噬细胞的吞噬率为99.81±0.13%,小鼠骨髓来源巨噬细胞的吞噬率为88.79±0.90%,结果表明裸鼹鼠巨噬细胞的吞噬功能强于小鼠巨噬细胞。polyi:c刺激巨噬细胞前后的比较相同浓度的polyi:c模拟病毒感染裸鼹鼠和小鼠骨髓来源巨噬细胞,一定时间后检测细胞的凋亡,分别提取rna和蛋白质,检测toll样受体3、干扰素β、nf-κb的表达量。结果表明,polyi:c刺激前后裸鼹鼠和小鼠骨髓来源巨噬细胞凋亡均有所升高;且细胞中tlr3、ifn-β、nf-κb的表达量均升高。相较于小鼠,裸鼹鼠骨髓来源巨噬细胞凋亡升高不显著,细胞中tlr3表达量升高不明显,但是ifn-β的表达却显著升高。lps刺激巨噬细胞前后的比较相同浓度的lps模拟细菌感染裸鼹鼠和小鼠骨髓来源巨噬细胞,一定时间后检测细胞的凋亡,分别提取rna和蛋白质,检测toll样受体4、肿瘤坏死因子α、核转录因子κb的表达量。结果表明,lps刺激前后裸鼹鼠骨髓来源巨噬细胞凋亡未有升高,而小鼠骨髓来源巨噬细胞凋亡率升高。细胞中tlr4、tnf-α、nf-κb的表达量均升高,相较于小鼠,裸鼹鼠骨髓来源tlr4蛋白表达量升高不显著,而细胞中nf-κb蛋白的表达量升高差异明显,且tnf-α的表达也显著升高。结论:采用流式细胞术证实裸鼹鼠脾脏中巨噬细胞的比例较高,继而进行裸鼹鼠和小鼠巨噬细胞功能的比较。流式细胞术检测结果表明裸鼹鼠巨噬细胞的吞噬功能强于小鼠巨噬细胞。采用荧光定量PCR和蛋白免疫印迹实验技术研究巨噬细胞产生免疫应答时可能存在的机制。病毒或细菌入侵时,裸鼹鼠巨噬细胞的TLRs被激活,进而启动NF-κB信号通路,从而引起细胞分泌各种细胞因子以抵抗外源物质的入侵。经过以上研究,我们推断在机体受到外源性抗原刺激时,裸鼹鼠巨噬细胞能够通过迅速吞噬外源异物并启动NF-κB信号通路产生大量的细胞因子快速产生免疫应答保护机体健康,该免疫应答机制这可能在其寿命长、自发肿瘤少有中具有重要作用。
[Abstract]:Background and objective: the naked mole rat is a year-round living in the underground, the rodent animal really social life, mainly in Africa and Kenya, Ethiopia and other regions in.1967, naked mole rats by foreign scholars introduced the laboratory, for half a century, the domestic and foreign scholars launched a series of research on the research report have been published in Nature, Science and other journals. In previous reports, the scholars at home and abroad to carry out research on the naked mole rat more concentrated in its long life, tolerance to hypoxia, lack of physical pain, spontaneous tumor rarely etc., but reports on its immune system is poorly understood. The immune system is an important index for biomedical research. Not only has the guiding significance for the judgment and the physiological state of animal feeding and management, is also an important basis for clinical and scientific research. The existing research report revealed between the system and the immune system of cancer The relationship with the aging of the body, the immune system has gradually deteriorated, this process is immune to aging, immune senescence and systemic inflammation, chronic inflammatory diseases and many cancers. Therefore, this was the first study on the immune system of naked mole rats were preliminarily studied, from the perspective of immunology to explain the life of naked mole rats a long, low rate of mechanism of biological characteristics of tumors, and human age-related diseases, provide a theoretical basis for the pathogenesis of inflammatory diseases and cancer, but also for the elderly disease, to provide new ideas and ideas for the treatment of inflammatory diseases and tumor diseases. Methods: This study selected a preliminary study on the immune cell macrophages body the most important immune organ spleen and body of the naked mole rat immune system. Firstly, the separation of naked mole rats and mouse spleen lymphocytes by flow cytometry, than Compared with naked mole rats and immune cells in the spleen of mice proportion. Secondly, methods to establish the naked mole rat bone marrow-derived macrophages were isolated and cultured, using flow cytometry to compare the phagocytosis of naked mole rats and mouse bone marrow-derived macrophages. Once again, the polyI:C simulation of virus infection of bone marrow-derived macrophages apoptosis by comparison before and after stimulation of naked mole rats and mouse bone marrow macrophages by flow cytometry, the fluorescent quantitative PCR technology comparison before and after stimulation of naked mole rats and mouse bone marrow-derived macrophages TLR3 and IFN- expression changes, further using Western blot technique before and after stimulation of naked mole rats and mouse bone marrow-derived macrophages TLR3 and NF- kappa B expression quantity, mechanism of naked mole rats macrophages may exist in resistance to virus invasion. Finally, LPS simulation of gram negative bacterial infection of bone marrow derived 宸ㄥ櫖缁嗚優,閲囩敤娴佸紡缁嗚優鏈�姣旇緝鍒烘浚�

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