流感病毒对人外周血γδT细胞早期激活及其交叉杀伤作用的研究

发布时间：2018-08-22 14:34

【摘要】：研究背景自1918年爆发第一次流感世界性大暴发流行开始，流感在载入历史的近100年历史中，共有4次世界性大流行，病造成上千万人感染和死亡，并给人们生活、健康和社会经济均造成严重影响。疫苗一直是人类防治流感病毒感染的经典方法，但流感病毒的快速变异给疫苗研制带来困难，寻找对高变异的流感病毒具有交叉保护的机制是防治流感病毒感染研究的热点和难点。目前流感疫苗研究主要集中在其激发的体液免疫，但一般缺乏对各亚型和新变异毒株的交叉保护作用。细胞免疫中，特异性T细胞能为各亚型病毒提供交叉反应从而起到保护作用，但目前研究证明能诱导产生记忆β T细胞的疫苗较难。Vγ9V2T细胞是人外周血中能特异的识别个别抗原的主要记忆细胞群，兼具固有和特异性免疫特性，有杀死微生物感染的细胞的能力，并且参与启动适应性免疫过程。其抗原识别阶段不受MHC限制的特点，能为研制具有交叉免疫效果的疫苗提供新的方向。近年来发现其能在体外扩增并溶解受感染的细胞，甲型流感病毒的各变异亚型能有效激活Vγ9V2T细胞，同时甲羟戊酸途径在甲型流感活化Vγ9V2T细胞中发挥一定左右，预示着γ T细胞对高变异病毒的潜在交叉免疫保护作用。然而这种潜在的交叉免疫保护机制如何？在其他型、亚型流感病毒中，γ T细胞交叉免疫保护作用的机制是否相同？目前仍处于不断加深认识的研究阶段。探明γ T细胞是否存在对异型病毒的广谱识别和交叉杀伤作用，并探讨病毒对γ T细胞激活的途径对于抗病毒防治工作和疫苗的研发有重要的作用。研究目的通过体外细胞实验探讨γ T细胞作为广谱抗流感病毒的免疫细胞的可能性。为研究和开发具有广谱抗流感病毒功效的治疗策略和通用疫苗提供基础科学依据。探讨流感病毒这类高变异RNA病毒被γ T细胞广谱识别和引起免疫应答的前期通路，为深入理解γ T细胞对流感病毒的识别和效应机制奠定基础，为抗流感治疗的药物研发提供新的靶点。研究方法 1.基于流式细胞技术的各型、亚型流感病毒对γ T细胞的刺激活化研究：采集10名健康自愿者外周血白膜，经蔗糖梯度密度离心法分离人外周血单个核细胞（PBMC），体外使用MOI（复感染系数）=2的流感病毒H1N1、H3N2、BV分别刺激PBMC细胞24小时后，流式细胞技术检测PBMC中总T细胞和γ T细胞的表面活化标志CD25和CD69以及细胞分化类型标志CD4和CD8。 2.基于乳酸脱氢酶释放试验（lactate dehydrogenase release test，LDH）的γ T细胞对流感病毒各型、亚型的杀伤作用研究：采集分离6名健康自愿者PBMC，体外使用rhGM-CSF（重组人粒-巨噬细胞集落刺激因子）刺激分化MDM（人外周血单核巨噬细胞）成熟，H1N1、H3N2、BV分别感染MDM成靶细胞，流感病毒刺激后PBMC经流式分选技术分选出的γ T细胞作为效应细胞，按效靶比10:1比例共同培养，LDH（乳酸脱氢酶释放试验）检测活化后γ T细胞对各流感病毒感染MDM的杀伤力和交叉杀伤能力。 3.甲羟戊酸途径对γ T细胞活化和杀伤作用的影响研究：采集分离10名健康自愿者PBMC，使用浓度为10μM的氟伐他汀前期作用PBMC4小时，后使用MOI=2的流感病毒H1N1、H3N2、BV分别体外刺激PBMC细胞，24小时后，流式细胞技术检测上述同类活化和分化指标。使用经氟伐他汀前期处理再病毒刺激的γ T细胞作为效应细胞，与流感病毒感染的MDM靶细胞按10:1比例培养，经LDH试验检测效应细胞对靶细胞的杀伤和交叉杀伤能力，探索甲羟戊酸途径对γ T细胞杀伤能力的影响。结果 1.正常人群中，T细胞和γ T细胞处于未活化状态，表现为细胞表面活化标志CD25、CD69和CD25+CD69+的低表达，其中T细胞该两标志的平均表达水平为9.76%、14.09%和2.79%，γ T细胞该两标志的平均表达水平为5.88%、8.46%和2.77%。正常人群中，T细胞和γ T细胞主要为CD4-CD8-状态（分别为31.48%和61.02%），CD4+CD8+的表达水平均处于低表达（1.00%和2.64%），表现为非效应状态的初始状态T细胞。 2. γ T细胞能经过各型、亚型流感病毒的刺激在24小时内得到早期活化，H1N1、H3N2、BV流感刺激后γ T细胞的CD25、CD69以及CD25+CD69+标志平均表达水平分别达到24.60%、40.11%、22.23%；17.79%、29.81%、15.24%；16.87%、29.84%、15.63%。活化后γ T细胞存在一定程度的CD4+CD8+功能型细胞6.22%、4.14%和3.48%。 3. H1N1、H3N2、BV流感病毒刺激γ T细胞24小时候，活化的γ T细胞对同一病毒感染的MDCK和MDM细胞系的杀伤水平分别达到43.59%、36.76%、21.59%和42.94%、38.15%、23.13%，均表现为较高的杀伤功效。甲型流感病毒活化的γ T细胞表现出较高的交叉杀伤功效，以H1N1刺激后的γ T细胞的交叉杀伤作用最强，对H3N2和BV感染MDM的杀伤作用平均分别达到36.99%和18.16%；H3N2活化的γ T细胞地交叉杀伤作用次之，，对H1N1和BV感染MDM的杀伤作用平均分别达到29.99%、10.63%。 4.氟伐他汀通过甲羟戊酸途径抑制流感病毒对γ T细胞的活化和功能型γ T细胞的杀伤作用，表现为经氟伐他汀前期处理γ T细胞，H1N1、H3N2、BV对γ T细胞的活化水平下降。其中使各H1N1实验组γ T细胞的CD25单阳、CD69单阳以及CD25、CD69双阳性活化标志较刺激前下降56.14%、57.71%和62.12%；H3N2组分别下降40.92%、57.63%和57.55%；BV组分别下降30.88%、39.24%和42.87%。直接杀伤作用H1N1、H3N2、BV各组分别下降42.77%、75.66%和60.63%；H1N1组对H3N2、BV感染靶细胞交叉杀伤作用分别下降67.96%和51.28%，H3N2组对H1N1、BV感染靶细胞交叉杀伤作用分别下降80.17%和56.52%，BV组对H1N1、H3N2感染靶细胞交叉杀伤作用分别下降10.06%和14.48%。结论 1.正常人群外周血中T细胞、γ T细胞处于未激活状态，细胞主要为CD4CD8双阴性类别，有部分CD8单阳性细胞，几乎没有CD4阳性细胞。 2.流感病毒能体外早期（24小时内）快速活化γ T细胞，且活化能力无型别、亚型间的差异；流感病毒刺激活化后γ T细胞有趋势分化成效应性T细胞，细胞主要向CD4单阳和CD4CD8双阳方向发展。γ T细胞的分化受型和亚型影响，以H1N1刺激γ T细胞向CD4CD8双阳性功能型细胞分化最为明显。 3.流感病毒刺激γ T细胞活化，开启γ T细胞特异性杀伤作用和交叉杀伤作用；且已甲型流感（H1N1、H3N2）刺激后表现明显。 4. γ T细胞的活化和杀伤作用受甲羟戊酸途径影响，甲羟戊酸途径极可能是流感病毒刺激活化γ T细胞的潜在途径。
[Abstract]:Research background
Since the first pandemic of influenza in 1918, there have been four pandemics in the history of nearly 100 years. The disease has caused tens of millions of infections and deaths, and has a serious impact on people's lives, health and socio-economic. Vaccines have been the classic method of human prevention and treatment of influenza virus infection. Rapid mutation of influenza virus makes it difficult to develop a vaccine. It is a hot and difficult problem to find a cross-protective mechanism for influenza virus with high mutation. In cellular immunity, specific T cells can provide cross-reactivity to various subtypes of viruses and thus play a protective role, but current studies have proved that it is difficult to induce a vaccine to produce memory beta T cells. In recent years, it has been found that it can amplify and dissolve infected cells in vitro. Variant subtypes of influenza A virus can activate V gamma effectively. 9V2T cells and mevalonate pathway play a certain role in influenza A-activated V 9V2T cells, indicating the potential cross-immune protection of gamma T cells against highly variable viruses.
However, what is the potential mechanism of cross-immune protection? Are the mechanisms of cross-immune protection of gamma T cells the same in other influenza viruses and subtypes? It is still in the stage of deepening understanding. It is necessary to find out if there are broad-spectrum recognition and cross-killing effects of gamma T cells on heterotypic viruses, and to explore the role of the virus in the detection of gamma T cells. Cell activation pathway plays an important role in antiviral control and vaccine research and development.
research objective
The possibility of using gamma T cells as broad-spectrum anti-influenza immune cells was investigated by cell experiments in vitro, which provided basic scientific basis for the research and development of therapeutic strategies and general vaccines with broad-spectrum anti-influenza efficacy.
To explore the early pathways through which highly variable RNA viruses such as influenza viruses are widely recognized by gamma T cells and induce immune responses, and to lay a foundation for further understanding the mechanism of recognition and effect of gamma T cells on influenza viruses, and to provide a new target for the development of anti-influenza drugs.
research method
1. Activation of human peripheral blood mononuclear cells (PBMCs) stimulated by influenza virus subtypes (H1N1, H3N2, BV) based on flow cytometry: 10 healthy volunteers'peripheral blood albumin membranes were collected and isolated by sucrose gradient density centrifugation. Flow cytometry was used to detect the surface activation markers CD25 and CD69 of total T and gamma T cells and the cell differentiation markers CD4 and CD8 in PBMC.
2. The lethal effect of gamma-T cells on influenza virus subtypes and subtypes based on lactate dehydrogenase release test (LDH): PBMCs from six healthy volunteers were collected and isolated, and rhGM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor) was used to stimulate the maturation of differentiated MDM (human peripheral blood mononuclear macrophage) in vitro, H1N1 H3N2 and BV infected MDM target cells respectively. After stimulation by influenza virus, PBMC selected gamma T cells were used as effector cells and co-cultured in the ratio of 10:1. LDH (lactate dehydrogenase release test) was used to detect the killing and cross-killing abilities of activated gamma T cells against influenza virus-infected MDM.
3. Effect of mevalonate pathway on activation and cytotoxicity of gamma T cells: PBMCs from 10 healthy volunteers were collected and isolated. Pretreatment with fluvastatin at a concentration of 10 mu M for 4 hours, and then stimulation of PBMC cells by influenza viruses H1N1, H3N2 and BV with MOI=2 were performed in vitro. Flow cytometry was used to detect the activation and fraction of the same kind of PBMC after 24 hours. Chemical indices. The effector cells were cultured in a 10:1 ratio with MDM target cells infected by influenza virus. The killing and cross-killing abilities of effector cells to target cells were tested by LDH assay to explore the effect of mevalonate pathway on killing abilities of gamma T cells.
Result
1. T cells and gamma T cells were inactivated in the normal population, showing a low expression of CD25, CD69 and CD25+CD69+ markers. The average expression levels of these two markers were 9.76%, 14.09% and 2.79% in T cells, and 5.88%, 8.46% and 2.77% in gamma T cells. CD4-CD8-state was the predominant (31.48% and 61.02% respectively). The expression level of CD4+CD8+ was low (1.00% and 2.64%) and showed the initial state of non-effect T cells.
2. The average expression levels of CD25, CD69 and CD25+CD69+ markers in gamma T cells stimulated by H1N1, H3N2 and BV influenza reached 24.60%, 40.11%, 22.23%, 17.79%, 29.81%, 15.24%, 16.87%, 29.84% and 15.63% respectively. After activation, the expression levels of CD25, CD69 and CD25+CD69+ markers in gamma T cells reached a certain extent. 4+CD8+ functional cells 6.22%, 4.14% and 3.48%.
3.When H1N1, H3N2 and BV influenza viruses stimulated gamma T cells for 24 hours, the killing levels of activated gamma T cells to MDCK and MDM cell lines infected by the same virus reached 43.59%, 36.76%, 21.59% and 42.94%, 38.15% and 23.13% respectively, which showed higher killing efficacy. H1N1-stimulated gamma T cells showed the strongest cross-killing effect, with an average of 36.99% and 18.16% against H3N2 and BV-infected MDM, followed by H3N2-activated gamma T cells, with an average of 29.99% and 10.63% against H1N1 and BV-infected MDM, respectively.
4. Fluvastatin inhibited the activation of influenza virus on gamma T cells and the killing effect of functional gamma T cells through methylolpyruvate pathway. The activation level of gamma T cells, H1N1, H3N2 and BV was decreased after fluvastatin pretreatment, and the activation of CD25, CD69, CD25 and CD69 of gamma T cells in each H1N1 experimental group was induced by fluvastatin. Signs decreased by 56.14%, 57.71% and 62.12%, H3N2 group by 40.92%, 57.63% and 57.55%, BV group by 30.88%, 39.24% and 42.87%, H1N1, H3N2 and BV groups by 42.77%, 75.66% and 60.63% respectively, H1N1 group by 67.96% and 51.28% respectively, H3N2 group by H1N1, BV infection target cells by cross-killing, H1N1, H3N2 and H1N1, BV infection target cells by 51.28% respectively. The cross-killing effect of BV infected target cells decreased by 80.17% and 56.52% respectively. The cross-killing effect of BV group on H1N1 and H3N2 infected target cells decreased by 10.06% and 14.48% respectively.
conclusion
1. The T cells and gamma T cells in the peripheral blood of normal people are in the inactivated state. The cells are mainly CD4 CD8-negative. Some CD8-positive cells are single positive, and almost no CD4-positive cells.
2. Influenza viruses can rapidly activate gamma T cells in vitro (within 24 hours), and the activation capacity is no type, subtype difference; after the activation of influenza viruses, gamma T cells tend to differentiate into effective T cells, cells mainly to CD4 single positive and CD4 CD8 double positive direction. The differentiation of gamma T cells is affected by type and subtype, and stimulated by H1N1 to gamma T cells. The differentiation of CD4CD8 double positive functional cells was most obvious.
3. Influenza viruses stimulate the activation of gamma T cells, opening up the specific killing and cross-killing effects of gamma T cells, and have been significantly stimulated by influenza A (H1N1, H3N2).
4. The activation and killing effects of gamma T cells are affected by the mevalonate pathway, which may be a potential pathway for influenza viruses to stimulate the activation of gamma T cells.
【学位授予单位】：中山大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R373.13

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