本文關(guān)鍵詞：A型流感病毒誘導(dǎo)外周血單個核細(xì)胞凋亡研究　出處：《汕頭大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

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【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傳染病,其起病急,蔓延快,對人群尤其是兒童、老年人及免疫缺陷者造成很大的危害。流感病毒點突變造成的抗原漂移可導(dǎo)致流感每年季節(jié)性流行,而基因重配造成的抗原轉(zhuǎn)換則可能產(chǎn)生新的亞型,因人類對新亞型普遍缺乏免疫力而可能導(dǎo)致流感世界大流行。 流感病毒已被證明能誘導(dǎo)各組織細(xì)胞的凋亡,包括外周血單個核細(xì)胞。已經(jīng)證明:從小鼠中分離得到的淋巴細(xì)胞能被從人身上分離得到的高致病A型流感病毒所誘導(dǎo),而導(dǎo)致凋亡的發(fā)生,但流感病毒誘導(dǎo)其凋亡的相關(guān)免疫病理學(xué)機制并沒得到進一步的闡述。研究表明,在病毒感染細(xì)胞時,病毒自身蛋白以及細(xì)胞分泌的信號分子都參與到這一過程中,比如,FAS-FASL、腫瘤壞死因子(TNF),RNA依賴的蛋白激酶R(PKR),氧化亞氮,轉(zhuǎn)化生長因子(TGF),有絲分裂原激活蛋白(MAP)激酶。盡管許多蛋白被證明與流感病毒的凋亡有關(guān),但并沒有一個統(tǒng)一的理論來概括各種因素。 在許多生理過程中,凋亡都是自發(fā)產(chǎn)生的,比如組織萎縮,免疫系統(tǒng)的發(fā)育等等。同樣的,凋亡在許多傳染性疾病的病理變化過程中也扮演著重要的角色,比如由病毒所引起的感染,盡管有許多的細(xì)胞內(nèi)蛋白牽涉到這一過程,但腫瘤抑制蛋白P53作為一種調(diào)節(jié)性蛋白,在腫瘤抑制、凋亡的調(diào)控、細(xì)胞周期的調(diào)節(jié)上都起著重要的作用。P53還是各種外界應(yīng)急壓力下抑制異常細(xì)胞過度生長的主要組分之一。各種細(xì)胞外界壓力,包括病毒感染,藥物處理等,均能激活P53的表達,進而導(dǎo)致細(xì)胞周期停滯或發(fā)生凋亡。P53的調(diào)節(jié)作用主要通過對蛋白磷酸化機制的穩(wěn)定,增強核定位,改變構(gòu)象,增強DNA的結(jié)合等幾個方面來起作用。 本實驗選用各不同健康人的外周血,從中分離得到淋巴細(xì)胞和單核巨噬細(xì)胞,并用H1N1 A型流感病毒感染,通過流式PI單染色以及Annexin V-PI雙染色檢測并比較外周血淋巴細(xì)胞以及單核巨噬細(xì)胞在不同情況下的凋亡情況。RT-PCR分別檢測P53,IFN的RNA表達水平。根據(jù)檢測的結(jié)果,加入了Pifithrin-α(PFT-α)抑制P53轉(zhuǎn)錄,檢測P53被抑制時,外周血淋巴細(xì)胞以及單核巨噬細(xì)胞的凋亡情況。 材料與方法 選取由本實驗室提供的A型流感病毒A/Shantou/169/2006(H1N1)1株,選用流感病毒分離鑒定細(xì)胞系MDCK(Madin-Darby canine kidney)細(xì)胞,通過紅細(xì)胞凝集試驗(Hemagglutination assay, HA)、空斑形成試驗(Plaque-forming assay),得到空斑形成單位(Plaque forming unit, PFU)為1.2×107 PFU/ml的病毒液。采集志愿者的新鮮血液并于1h內(nèi)用Ficoll淋巴細(xì)胞分離液分離并得到單個核細(xì)胞、淋巴細(xì)胞以及單核巨噬細(xì)胞。用病毒液吸附不同處理方式的細(xì)胞:包括1.病毒直接吸附單個核細(xì)胞;2.病毒直接吸附淋巴細(xì)胞以及單核巨噬細(xì)胞1h后,再將淋巴細(xì)胞和單核巨噬細(xì)胞進行分離;3.將淋巴細(xì)胞,單核巨噬細(xì)胞進行分離后再用流感病毒進行吸附;4.病毒吸附不同時間后,淋巴細(xì)胞、單核巨噬細(xì)胞的凋亡情況�？刂聘腥緩�(fù)數(shù)(Multiplicity of infection, MOI)為2。并使用流式細(xì)胞術(shù),通過碘化丙錠(Propiolium, PI)單染色和Annexin V-PI雙染色的方法檢測細(xì)胞的凋亡,以確認(rèn)不同處理間凋亡是否存在差異。同時,提取病毒感染后培養(yǎng)2h,4h,8h,12h淋巴細(xì)胞以及單核巨噬細(xì)胞的RNA,檢測淋巴細(xì)胞,單核巨噬細(xì)胞中P53,IFN在RNA表達水平上的差異。根據(jù)PCR結(jié)果,使用Pifithrin-α(PFT-α)抑制P53的轉(zhuǎn)錄,并通過流式PI單染色檢測淋巴細(xì)胞、單核巨噬細(xì)胞的凋亡情況。 結(jié)果 1.所取H1N1 A型流感病毒在MDCK中進行培養(yǎng)后,滴度有所下降,滴度從512下降到培養(yǎng)后的256. 2.空斑形成試驗:PFU=1.2×107 PFU/ml 3. PI染色結(jié)果:淋巴細(xì)胞在病毒的作用下凋亡率逐漸增加,但同時隨著淋巴細(xì)胞體外培養(yǎng)時間的延長,淋巴細(xì)胞自身出現(xiàn)的凋亡也逐漸增加。在24h,加入流感病毒的單核巨噬細(xì)胞其凋亡較未加入病毒的對照組要高,而從48h開始,加入病毒的單核巨噬細(xì)胞其凋亡則反較對照組要低。Annexin V-PI雙染色結(jié)果與PI單染色結(jié)果一致。 4.將淋巴細(xì)胞、單核巨噬細(xì)胞用流感病毒吸附1h后再進行分離與淋巴細(xì)胞、單核巨噬細(xì)胞分離后再進行病毒吸附1h,培養(yǎng)48h后分別進行凋亡檢測,其中淋巴細(xì)胞凋亡比例前者較后者要低。 5.熒光染色結(jié)果:流感病毒分別吸附1h,2h,4h,8h,培養(yǎng)48h后,各凋亡比例間不存在差異。 6. RT-PCR結(jié)果:淋巴細(xì)胞在2h,4h,P53表達水平均升高,對照組在8h ,P53仍有少量表達。單核巨噬細(xì)胞在4h后,病毒感染組,其P53表達水平要明顯升高。IFN RT-PCR結(jié)果無差異。 7.加入抑制劑PFT-α后PI染色:淋巴細(xì)胞,單核巨噬細(xì)胞凋亡峰均消失,凋亡被抑制。 結(jié)論 1.流感病毒分別感染單個核細(xì)胞,淋巴細(xì)胞,單核巨噬細(xì)胞,其各自的凋亡比例之間的差異顯示了在流感病毒誘導(dǎo)的凋亡中,淋巴細(xì)胞與單核巨噬細(xì)胞之間是存在相互作用的。 2.流感病毒在細(xì)胞中吸附不同時間后,檢測細(xì)胞凋亡情況,說明凋亡與流感病毒吸附時間的長短不存在相關(guān)性。 3.流感病毒能夠誘導(dǎo)淋巴細(xì)胞凋亡,而對單核巨噬細(xì)胞,病毒吸附后,培養(yǎng)48h前表現(xiàn)為誘導(dǎo)凋亡,培養(yǎng)48h后則表現(xiàn)為抑制凋亡,在不同的時間段表現(xiàn)為不同的效果。 4.通過對IFN,P53 RNA水平的檢測結(jié)果,說明流感病毒的凋亡過程可能沒有IFN的直接參與,而P53則可能參與到了淋巴細(xì)胞或單核巨噬細(xì)胞的凋亡過程。 5.加入P53抑制劑后,流式PI單染色結(jié)果顯示,凋亡明顯下降,說明P53可能在H1N1流感病毒所誘導(dǎo)的淋巴細(xì)胞和單核巨噬細(xì)胞的起到促凋亡中進作用。
[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.

【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R373

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相關(guān)期刊論文 前1條

1 朱建國;華修國;艾曉杰;;A型流感病毒跨動物種感染機制的研究進展[J];中國獸醫(yī)學(xué)報;2006年05期

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本文編號：1413565