發(fā)布時間：2018-03-21 15:17

  本文選題：裸鼴鼠　切入點：小鼠　出處：《第二軍醫(yī)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景及目的:裸鼴鼠是一種終年生活在地下、營真社會性生活的嚙齒類動物,多分布于非洲肯尼亞、埃塞俄比亞等地區(qū)。1967年,裸鼴鼠被國外學(xué)者引進(jìn)實驗室,半個世紀(jì)以來,國內(nèi)外學(xué)者對其展開了一系列的研究,研究報道相繼發(fā)表于Nature、Science等期刊。在已有的報道中,國內(nèi)外學(xué)者針對裸鼴鼠開展的研究多集中于其壽命長、耐受低氧、缺少疼痛物質(zhì)、自發(fā)性腫瘤鮮有等方面,但是對其免疫系統(tǒng)的研究報道甚少。免疫系統(tǒng)是生物醫(yī)學(xué)研究的重要指標(biāo),不僅對動物生理狀態(tài)的判斷和飼養(yǎng)管理具有指導(dǎo)意義,也是臨床醫(yī)療和科學(xué)研究的重要依據(jù)。目前已有研究報告揭示免疫系統(tǒng)與癌癥之間的關(guān)系:隨著機(jī)體的衰老,其免疫系統(tǒng)也逐漸惡化,這個過程即為免疫衰老,而免疫衰老與全身性炎癥、慢性炎癥性疾病以及許多癌癥相關(guān)。因此,本研究率先針對裸鼴鼠的免疫系統(tǒng)進(jìn)行初步研究,從免疫學(xué)的角度來闡釋裸鼴鼠壽命長、腫瘤發(fā)生率低等生物學(xué)特性的機(jī)制,進(jìn)而為人類老年疾病、炎癥性疾病及腫瘤的發(fā)病機(jī)制提供理論依據(jù),同時也為老年疾病、炎癥性疾病及腫瘤等疾病的治療提供新的思路和想法。研究方法:本研究分別選取機(jī)體最大的免疫器官脾臟和機(jī)體重要的免疫細(xì)胞巨噬細(xì)胞進(jìn)行裸鼴鼠免疫系統(tǒng)的初步研究。首先,分離裸鼴鼠和小鼠脾臟淋巴細(xì)胞,采用流式細(xì)胞術(shù)比較裸鼴鼠和小鼠脾臟中免疫細(xì)胞的比例。其次,建立裸鼴鼠骨髓來源巨噬細(xì)胞分離培養(yǎng)的方法,采用流式細(xì)胞術(shù)比較裸鼴鼠和小鼠骨髓來源巨噬細(xì)胞的吞噬功能。再次,polyI:C模擬病毒感染骨髓來源巨噬細(xì)胞,采用流式細(xì)胞術(shù)比較刺激前后裸鼴鼠和小鼠骨髓來源巨噬細(xì)胞的凋亡情況,采用熒光定量PCR技術(shù)比較刺激前后裸鼴鼠和小鼠骨髓來源巨噬細(xì)胞TLR3和IFN-β表達(dá)量的變化情況,進(jìn)一步采用蛋白免疫印跡技術(shù)比較刺激前后裸鼴鼠和小鼠骨髓來源巨噬細(xì)胞TLR3和NF-κB表達(dá)量的變化情況,研究裸鼴鼠巨噬細(xì)胞在抵抗病毒入侵中可能存在的機(jī)制。最后,LPS模擬革蘭氏陰性細(xì)菌感染骨髓來源巨噬細(xì)胞,采用流式細(xì)胞術(shù)比較刺激前后裸鼴鼠和小鼠骨髓來源巨噬細(xì)胞的凋亡情況,采用熒光定量PCR技術(shù)比較刺激前后裸鼴鼠和小鼠骨髓來源巨噬細(xì)胞TLR4和TNF-α表達(dá)量的變化情況,進(jìn)一步采用蛋白免疫印跡技術(shù)比較刺激前后裸鼴鼠和小鼠骨髓來源巨噬細(xì)胞TLR4和NF-κB表達(dá)量的變化情況,研究裸鼴鼠巨噬細(xì)胞在抵抗細(xì)菌感染中可能存在的機(jī)制。結(jié)果:脾臟淋巴細(xì)胞中免疫細(xì)胞比例比較分離裸鼴鼠和小鼠脾臟淋巴細(xì)胞,采用流式細(xì)胞術(shù)檢測各類免疫細(xì)胞的比例,結(jié)果如下:小鼠脾臟巨噬細(xì)胞的比例為15.54±2.12%,b細(xì)胞的比例為43.21±9.34%,t細(xì)胞的比例為37.54±4.50%,nk細(xì)胞的比例為6.06±0.23%;裸鼴鼠脾臟巨噬細(xì)胞的比例為34.96±5.85%,b細(xì)胞的比例為2.34±0.67%,t細(xì)胞的比例為32.14±4.66%,nk細(xì)胞的比例為3.09±0.16%。和小鼠相比較,裸鼴鼠脾臟淋巴細(xì)胞中巨噬細(xì)胞的比例較高,而b細(xì)胞的比例較低,t細(xì)胞和nk細(xì)胞的比例基本無差異。建立骨髓來源巨噬細(xì)胞分離培養(yǎng)的方法及吞噬功能比較參照經(jīng)典的小鼠骨髓來源巨噬細(xì)胞分離培養(yǎng)的方法,首先分離裸鼴鼠骨髓干細(xì)胞,然后利用粒細(xì)胞-巨噬細(xì)胞集落刺激因子誘導(dǎo)造血干細(xì)胞轉(zhuǎn)化為巨噬細(xì)胞,并采用流式細(xì)胞術(shù)進(jìn)行純度的鑒定,檢測細(xì)胞表面蛋白cd11b比例高達(dá)90%,鑒定分離培養(yǎng)的細(xì)胞為骨髓來源巨噬細(xì)胞。分別采用吞噬紅細(xì)胞法和流式細(xì)胞術(shù)檢測裸鼴鼠和小鼠骨髓來源巨噬細(xì)胞的吞噬率,裸鼴鼠骨髓來源巨噬細(xì)胞的吞噬率為99.81±0.13%,小鼠骨髓來源巨噬細(xì)胞的吞噬率為88.79±0.90%,結(jié)果表明裸鼴鼠巨噬細(xì)胞的吞噬功能強(qiáng)于小鼠巨噬細(xì)胞。polyi:c刺激巨噬細(xì)胞前后的比較相同濃度的polyi:c模擬病毒感染裸鼴鼠和小鼠骨髓來源巨噬細(xì)胞,一定時間后檢測細(xì)胞的凋亡,分別提取rna和蛋白質(zhì),檢測toll樣受體3、干擾素β、nf-κb的表達(dá)量。結(jié)果表明,polyi:c刺激前后裸鼴鼠和小鼠骨髓來源巨噬細(xì)胞凋亡均有所升高;且細(xì)胞中tlr3、ifn-β、nf-κb的表達(dá)量均升高。相較于小鼠,裸鼴鼠骨髓來源巨噬細(xì)胞凋亡升高不顯著,細(xì)胞中tlr3表達(dá)量升高不明顯,但是ifn-β的表達(dá)卻顯著升高。lps刺激巨噬細(xì)胞前后的比較相同濃度的lps模擬細(xì)菌感染裸鼴鼠和小鼠骨髓來源巨噬細(xì)胞,一定時間后檢測細(xì)胞的凋亡,分別提取rna和蛋白質(zhì),檢測toll樣受體4、腫瘤壞死因子α、核轉(zhuǎn)錄因子κb的表達(dá)量。結(jié)果表明,lps刺激前后裸鼴鼠骨髓來源巨噬細(xì)胞凋亡未有升高,而小鼠骨髓來源巨噬細(xì)胞凋亡率升高。細(xì)胞中tlr4、tnf-α、nf-κb的表達(dá)量均升高,相較于小鼠,裸鼴鼠骨髓來源tlr4蛋白表達(dá)量升高不顯著,而細(xì)胞中nf-κb蛋白的表達(dá)量升高差異明顯,且tnf-α的表達(dá)也顯著升高。結(jié)論:采用流式細(xì)胞術(shù)證實裸鼴鼠脾臟中巨噬細(xì)胞的比例較高,繼而進(jìn)行裸鼴鼠和小鼠巨噬細(xì)胞功能的比較。流式細(xì)胞術(shù)檢測結(jié)果表明裸鼴鼠巨噬細(xì)胞的吞噬功能強(qiáng)于小鼠巨噬細(xì)胞。采用熒光定量PCR和蛋白免疫印跡實驗技術(shù)研究巨噬細(xì)胞產(chǎn)生免疫應(yīng)答時可能存在的機(jī)制。病毒或細(xì)菌入侵時,裸鼴鼠巨噬細(xì)胞的TLRs被激活,進(jìn)而啟動NF-κB信號通路,從而引起細(xì)胞分泌各種細(xì)胞因子以抵抗外源物質(zhì)的入侵。經(jīng)過以上研究,我們推斷在機(jī)體受到外源性抗原刺激時,裸鼴鼠巨噬細(xì)胞能夠通過迅速吞噬外源異物并啟動NF-κB信號通路產(chǎn)生大量的細(xì)胞因子快速產(chǎn)生免疫應(yīng)答保護(hù)機(jī)體健康,該免疫應(yīng)答機(jī)制這可能在其壽命長、自發(fā)腫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櫖緇嗚優(yōu),閲囩敤嫻佸紡緇嗚優(yōu)鏈瘮杈冨埡嬋，

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