A型流感病毒诱导外周血单个核细胞凋亡研究

发布时间：2018-01-12 09:03

本文关键词：A型流感病毒诱导外周血单个核细胞凋亡研究　出处：《汕头大学》2008年硕士论文　论文类型：学位论文

【摘要】： 背景及目的流行性感冒是由流感病毒引起的急性呼吸道传染病,其起病急,蔓延快,对人群尤其是儿童、老年人及免疫缺陷者造成很大的危害。流感病毒点突变造成的抗原漂移可导致流感每年季节性流行,而基因重配造成的抗原转换则可能产生新的亚型,因人类对新亚型普遍缺乏免疫力而可能导致流感世界大流行。流感病毒已被证明能诱导各组织细胞的凋亡,包括外周血单个核细胞。已经证明:从小鼠中分离得到的淋巴细胞能被从人身上分离得到的高致病A型流感病毒所诱导,而导致凋亡的发生,但流感病毒诱导其凋亡的相关免疫病理学机制并没得到进一步的阐述。研究表明,在病毒感染细胞时,病毒自身蛋白以及细胞分泌的信号分子都参与到这一过程中,比如,FAS-FASL、肿瘤坏死因子(TNF),RNA依赖的蛋白激酶R(PKR),氧化亚氮,转化生长因子(TGF),有丝分裂原激活蛋白(MAP)激酶。尽管许多蛋白被证明与流感病毒的凋亡有关,但并没有一个统一的理论来概括各种因素。在许多生理过程中,凋亡都是自发产生的,比如组织萎缩,免疫系统的发育等等。同样的,凋亡在许多传染性疾病的病理变化过程中也扮演着重要的角色,比如由病毒所引起的感染,尽管有许多的细胞内蛋白牵涉到这一过程,但肿瘤抑制蛋白P53作为一种调节性蛋白,在肿瘤抑制、凋亡的调控、细胞周期的调节上都起着重要的作用。P53还是各种外界应急压力下抑制异常细胞过度生长的主要组分之一。各种细胞外界压力,包括病毒感染,药物处理等,均能激活P53的表达,进而导致细胞周期停滞或发生凋亡。P53的调节作用主要通过对蛋白磷酸化机制的稳定,增强核定位,改变构象,增强DNA的结合等几个方面来起作用。本实验选用各不同健康人的外周血,从中分离得到淋巴细胞和单核巨噬细胞,并用H1N1 A型流感病毒感染,通过流式PI单染色以及Annexin V-PI双染色检测并比较外周血淋巴细胞以及单核巨噬细胞在不同情况下的凋亡情况。RT-PCR分别检测P53,IFN的RNA表达水平。根据检测的结果,加入了Pifithrin-α(PFT-α)抑制P53转录,检测P53被抑制时,外周血淋巴细胞以及单核巨噬细胞的凋亡情况。材料与方法选取由本实验室提供的A型流感病毒A/Shantou/169/2006(H1N1)1株,选用流感病毒分离鉴定细胞系MDCK(Madin-Darby canine kidney)细胞,通过红细胞凝集试验(Hemagglutination assay, HA)、空斑形成试验(Plaque-forming assay),得到空斑形成单位(Plaque forming unit, PFU)为1.2×107 PFU/ml的病毒液。采集志愿者的新鲜血液并于1h内用Ficoll淋巴细胞分离液分离并得到单个核细胞、淋巴细胞以及单核巨噬细胞。用病毒液吸附不同处理方式的细胞:包括1.病毒直接吸附单个核细胞;2.病毒直接吸附淋巴细胞以及单核巨噬细胞1h后,再将淋巴细胞和单核巨噬细胞进行分离;3.将淋巴细胞,单核巨噬细胞进行分离后再用流感病毒进行吸附;4.病毒吸附不同时间后,淋巴细胞、单核巨噬细胞的凋亡情况。控制感染复数(Multiplicity of infection, MOI)为2。并使用流式细胞术,通过碘化丙锭(Propiolium, PI)单染色和Annexin V-PI双染色的方法检测细胞的凋亡,以确认不同处理间凋亡是否存在差异。同时,提取病毒感染后培养2h,4h,8h,12h淋巴细胞以及单核巨噬细胞的RNA,检测淋巴细胞,单核巨噬细胞中P53,IFN在RNA表达水平上的差异。根据PCR结果,使用Pifithrin-α(PFT-α)抑制P53的转录,并通过流式PI单染色检测淋巴细胞、单核巨噬细胞的凋亡情况。结果 1.所取H1N1 A型流感病毒在MDCK中进行培养后,滴度有所下降,滴度从512下降到培养后的256. 2.空斑形成试验:PFU=1.2×107 PFU/ml 3. PI染色结果:淋巴细胞在病毒的作用下凋亡率逐渐增加,但同时随着淋巴细胞体外培养时间的延长,淋巴细胞自身出现的凋亡也逐渐增加。在24h,加入流感病毒的单核巨噬细胞其凋亡较未加入病毒的对照组要高,而从48h开始,加入病毒的单核巨噬细胞其凋亡则反较对照组要低。Annexin V-PI双染色结果与PI单染色结果一致。 4.将淋巴细胞、单核巨噬细胞用流感病毒吸附1h后再进行分离与淋巴细胞、单核巨噬细胞分离后再进行病毒吸附1h,培养48h后分别进行凋亡检测,其中淋巴细胞凋亡比例前者较后者要低。 5.荧光染色结果:流感病毒分别吸附1h,2h,4h,8h,培养48h后,各凋亡比例间不存在差异。 6. RT-PCR结果:淋巴细胞在2h,4h,P53表达水平均升高,对照组在8h ,P53仍有少量表达。单核巨噬细胞在4h后,病毒感染组,其P53表达水平要明显升高。IFN RT-PCR结果无差异。 7.加入抑制剂PFT-α后PI染色:淋巴细胞,单核巨噬细胞凋亡峰均消失,凋亡被抑制。结论 1.流感病毒分别感染单个核细胞,淋巴细胞,单核巨噬细胞,其各自的凋亡比例之间的差异显示了在流感病毒诱导的凋亡中,淋巴细胞与单核巨噬细胞之间是存在相互作用的。 2.流感病毒在细胞中吸附不同时间后,检测细胞凋亡情况,说明凋亡与流感病毒吸附时间的长短不存在相关性。 3.流感病毒能够诱导淋巴细胞凋亡,而对单核巨噬细胞,病毒吸附后,培养48h前表现为诱导凋亡,培养48h后则表现为抑制凋亡,在不同的时间段表现为不同的效果。 4.通过对IFN,P53 RNA水平的检测结果,说明流感病毒的凋亡过程可能没有IFN的直接参与,而P53则可能参与到了淋巴细胞或单核巨噬细胞的凋亡过程。 5.加入P53抑制剂后,流式PI单染色结果显示,凋亡明显下降,说明P53可能在H1N1流感病毒所诱导的淋巴细胞和单核巨噬细胞的起到促凋亡中进作用。
[Abstract]:Background and objective: influenza is an acute respiratory infectious disease caused by influenza virus, its rapid onset, spread fast, to people especially children, great harm caused by the elderly and immunocompromised. Influenza virus antigen drift caused by point mutations can lead to the annual seasonal flu epidemic, and gene reassortment caused by antigen conversion it may produce a new subtype, which may lead to a pandemic because of a general lack of immunity to human subtype.
The flu virus has been shown can induce apoptosis of cells in tissues, including peripheral blood mononuclear cells. Have proved that the isolated lymphocytes can be isolated from humans are highly pathogenic influenza virus type A from mice induced to apoptosis, immune related pathology but the influenza virus induced the mechanism of apoptosis has not been further elaborated. The study shows that in infected cells, the virus protein secreted signaling molecules and cell are involved in this process, for example, FAS-FASL, tumor necrosis factor (TNF), RNA dependent protein kinase R (PKR), Nitrous Oxide, transforming growth factor (TGF), mitogen activated protein kinase (MAP). Although many proteins proved apoptosis and influenza virus, but does not have a unified theory to summarize the various factors.
In many physiological processes, apoptosis is spontaneous, such as tissue atrophy, the development of the immune system and so on. Similarly, apoptosis plays an important role in the pathological process of many infectious diseases, such as caused by a virus infection, although there are many intracellular proteins involved in this process. But the tumor suppressor protein P53 as a regulatory protein in tumor suppression, apoptosis, cell cycle regulation plays an important role in.P53 or external emergency pressure inhibit abnormal cell overgrowth of the main component of the external pressure. A variety of cells, including viral infection, drug treatment, expression can activate P53, which led to the regulation of cell cycle arrest or apoptosis of.P53 by protein phosphorylation mechanism stability, enhanced nuclear localization, conformational change, enhanced DNA binding etc. Several aspects work.
In this experiment, the peripheral blood of healthy people are different, lymphocytes and macrophages isolated from H1N1, and A influenza virus infection by flow cytometry, PI staining and Annexin V-PI staining were detected in peripheral blood lymphocytes and mononuclear macrophages apoptosis of.RT-PCR in different conditions were detected P53, the expression level of IFN RNA. According to the test result, joined the Pifithrin- alpha (PFT- alpha) inhibit the transcription of P53, detection of P53 was inhibited, the apoptosis of peripheral blood lymphocytes and mononuclear macrophages.
Materials and methods
Select the A type influenza virus A/Shantou/169/2006 provided by our laboratory (H1N1) of 1 strains, the influenza virus isolation and identification of cell line MDCK (Madin-Darby canine kidney) cells, the erythrocyte agglutination test (Hemagglutination assay, HA), plaque formation test (Plaque-forming assay), plaque forming units (Plaque forming unit PFU get for the 1.2 * 107) virus was PFU/ml. The fresh blood collection and volunteers in the 1H by Ficoll lymphocyte separating medium and mononuclear cells, lymphocytes and macrophages. The adsorption of different treatments of cells with virus solution: including 1. virus direct adsorption of mononuclear cells; 2. virus directly adsorbed lymphocytes and monocyte macrophages after 1h, then lymphocytes and macrophages were isolated; 3. lymphocytes, monocytes and macrophages were isolated after influenza virus. Attached; 4. different time after virus adsorption, lymphocyte, apoptosis of mononuclear macrophage. Infection control (Multiplicity of infection, plural MOI) is 2. and the use of flow cytometry by propidium iodide (Propiolium, PI) to detect the apoptosis of single staining and Annexin V-PI double staining method, to confirm the different whether there are differences between treatment of apoptosis. At the same time, culture 2h, extraction of virus infection 4h, 8h, 12h lymphocytes and mononuclear macrophage RNA lymphocyte, macrophage, P53, IFN at the RNA expression level difference. According to the results of PCR, Pifithrin- alpha (PFT- alpha) transcription inhibition of P53. And through flow cytometry PI single staining was used to detect the apoptosis of lymphocytes, monocytes and macrophages.
Result
After 1. H1N1 A influenza viruses were cultured in MDCK, the titer decreased and the titer dropped from 512 to 256. after the culture.
2. plaque formation test: PFU=1.2 x 107 PFU/ml
3. PI staining results: lymphocytes in the virus under the action of the apoptosis rate increased gradually, but with the extension of time in vitro cultured lymphocytes, apoptosis of lymphocytes itself is also increasing. In the 24h control group, mononuclear macrophage apoptosis is adding influenza virus without virus must be high, and starting from 48h, single macrophage apoptosis, anti virus added to low.Annexin compared with the control group, V-PI double staining and PI staining results.
4., lymphocytes and mononuclear macrophages were adsorbed 1H by influenza virus. Then they were separated from lymphocytes and mononuclear macrophages. Then the virus was adsorbed on 1H. After cultured 48h, apoptosis was detected respectively, and the proportion of lymphocyte apoptosis was lower than that of the latter.
5. fluorescence staining results: influenza viruses adsorb 1H, 2h, 4h, and 8h respectively, and there is no difference in the percentage of apoptosis after the culture of 48h.
6. RT-PCR results: the expression level of lymphocytes in 2H, 4H and P53 increased. In control group, 8h and P53 still had a small amount of expression. After mononuclear macrophages were infected with 4h, the P53 expression level of virus infected group increased significantly,.IFN RT-PCR had no difference.
7. after adding inhibitor PFT- alpha to PI staining, the apoptotic peak of mononuclear macrophages disappeared and the apoptosis was inhibited.
conclusion
1., influenza viruses infected mononuclear cells, lymphocytes and mononuclear macrophages respectively. The difference between their respective apoptosis proportions showed that there was interaction between lymphocytes and mononuclear macrophages in influenza virus induced apoptosis.
After the 2. influenza virus was adsorbed for different time in the cell, the apoptosis was detected, indicating that there was no correlation between the apoptosis and the length of the influenza virus adsorption time.
3., influenza virus can induce apoptosis of lymphocytes, while monocytes macrophages and virus adsorbed, induce apoptosis before cultured 48h. After 48h culture, it shows inhibition of apoptosis, showing different effects in different time periods.
4., by detecting the level of IFN and P53 RNA, we can see that the apoptosis process of influenza virus may not be directly involved in IFN, while P53 may be involved in the apoptosis process of lymphocytes or monocytes.
5. after adding P53 inhibitor, flow cytometric PI staining showed that apoptosis decreased significantly, indicating that P53 may play an important role in promoting apoptosis of lymphocytes and monocytes macrophages induced by H1N1 influenza virus.

【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373

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