【摘要】：研究背景 自1918年爆發(fā)第一次流感世界性大暴發(fā)流行開始，流感在載入歷史的近100年歷史中，共有4次世界性大流行，病造成上千萬人感染和死亡，并給人們生活、健康和社會(huì)經(jīng)濟(jì)均造成嚴(yán)重影響。疫苗一直是人類防治流感病毒感染的經(jīng)典方法，但流感病毒的快速變異給疫苗研制帶來困難，尋找對(duì)高變異的流感病毒具有交叉保護(hù)的機(jī)制是防治流感病毒感染研究的熱點(diǎn)和難點(diǎn)。目前流感疫苗研究主要集中在其激發(fā)的體液免疫，但一般缺乏對(duì)各亞型和新變異毒株的交叉保護(hù)作用。細(xì)胞免疫中，特異性T細(xì)胞能為各亞型病毒提供交叉反應(yīng)從而起到保護(hù)作用，但目前研究證明能誘導(dǎo)產(chǎn)生記憶β T細(xì)胞的疫苗較難。Vγ9V2T細(xì)胞是人外周血中能特異的識(shí)別個(gè)別抗原的主要記憶細(xì)胞群，兼具固有和特異性免疫特性，有殺死微生物感染的細(xì)胞的能力，并且參與啟動(dòng)適應(yīng)性免疫過程。其抗原識(shí)別階段不受MHC限制的特點(diǎn)，能為研制具有交叉免疫效果的疫苗提供新的方向。近年來發(fā)現(xiàn)其能在體外擴(kuò)增并溶解受感染的細(xì)胞，甲型流感病毒的各變異亞型能有效激活Vγ9V2T細(xì)胞，同時(shí)甲羥戊酸途徑在甲型流感活化Vγ9V2T細(xì)胞中發(fā)揮一定左右，預(yù)示著γ T細(xì)胞對(duì)高變異病毒的潛在交叉免疫保護(hù)作用。 然而這種潛在的交叉免疫保護(hù)機(jī)制如何？在其他型、亞型流感病毒中，γ T細(xì)胞交叉免疫保護(hù)作用的機(jī)制是否相同？目前仍處于不斷加深認(rèn)識(shí)的研究階段。探明γ T細(xì)胞是否存在對(duì)異型病毒的廣譜識(shí)別和交叉殺傷作用，并探討病毒對(duì)γ T細(xì)胞激活的途徑對(duì)于抗病毒防治工作和疫苗的研發(fā)有重要的作用。 研究目的 通過體外細(xì)胞實(shí)驗(yàn)探討γ T細(xì)胞作為廣譜抗流感病毒的免疫細(xì)胞的可能性。為研究和開發(fā)具有廣譜抗流感病毒功效的治療策略和通用疫苗提供基礎(chǔ)科學(xué)依據(jù)。 探討流感病毒這類高變異RNA病毒被γ T細(xì)胞廣譜識(shí)別和引起免疫應(yīng)答的前期通路，為深入理解γ T細(xì)胞對(duì)流感病毒的識(shí)別和效應(yīng)機(jī)制奠定基礎(chǔ)，為抗流感治療的藥物研發(fā)提供新的靶點(diǎn)。 研究方法 1.基于流式細(xì)胞技術(shù)的各型、亞型流感病毒對(duì)γ T細(xì)胞的刺激活化研究：采集10名健康自愿者外周血白膜，經(jīng)蔗糖梯度密度離心法分離人外周血單個(gè)核細(xì)胞（PBMC），體外使用MOI（復(fù)感染系數(shù)）=2的流感病毒H1N1、H3N2、BV分別刺激PBMC細(xì)胞24小時(shí)后，流式細(xì)胞技術(shù)檢測PBMC中總T細(xì)胞和γ T細(xì)胞的表面活化標(biāo)志CD25和CD69以及細(xì)胞分化類型標(biāo)志CD4和CD8。 2.基于乳酸脫氫酶釋放試驗(yàn)（lactate dehydrogenase release test，LDH）的γ T細(xì)胞對(duì)流感病毒各型、亞型的殺傷作用研究：采集分離6名健康自愿者PBMC，體外使用rhGM-CSF（重組人粒-巨噬細(xì)胞集落刺激因子）刺激分化MDM（人外周血單核巨噬細(xì)胞）成熟，H1N1、H3N2、BV分別感染MDM成靶細(xì)胞，流感病毒刺激后PBMC經(jīng)流式分選技術(shù)分選出的γ T細(xì)胞作為效應(yīng)細(xì)胞，按效靶比10:1比例共同培養(yǎng)，LDH（乳酸脫氫酶釋放試驗(yàn)）檢測活化后γ T細(xì)胞對(duì)各流感病毒感染MDM的殺傷力和交叉殺傷能力。 3.甲羥戊酸途徑對(duì)γ T細(xì)胞活化和殺傷作用的影響研究：采集分離10名健康自愿者PBMC，使用濃度為10μM的氟伐他汀前期作用PBMC4小時(shí)，后使用MOI=2的流感病毒H1N1、H3N2、BV分別體外刺激PBMC細(xì)胞，24小時(shí)后，流式細(xì)胞技術(shù)檢測上述同類活化和分化指標(biāo)。使用經(jīng)氟伐他汀前期處理再病毒刺激的γ T細(xì)胞作為效應(yīng)細(xì)胞，與流感病毒感染的MDM靶細(xì)胞按10:1比例培養(yǎng)，經(jīng)LDH試驗(yàn)檢測效應(yīng)細(xì)胞對(duì)靶細(xì)胞的殺傷和交叉殺傷能力，探索甲羥戊酸途徑對(duì)γ T細(xì)胞殺傷能力的影響。 結(jié)果 1.正常人群中，T細(xì)胞和γ T細(xì)胞處于未活化狀態(tài)，表現(xiàn)為細(xì)胞表面活化標(biāo)志CD25、CD69和CD25+CD69+的低表達(dá)，其中T細(xì)胞該兩標(biāo)志的平均表達(dá)水平為9.76%、14.09%和2.79%，γ T細(xì)胞該兩標(biāo)志的平均表達(dá)水平為5.88%、8.46%和2.77%。正常人群中，T細(xì)胞和γ T細(xì)胞主要為CD4-CD8-狀態(tài)（分別為31.48%和61.02%），CD4+CD8+的表達(dá)水平均處于低表達(dá)（1.00%和2.64%），表現(xiàn)為非效應(yīng)狀態(tài)的初始狀態(tài)T細(xì)胞。 2. γ T細(xì)胞能經(jīng)過各型、亞型流感病毒的刺激在24小時(shí)內(nèi)得到早期活化，H1N1、H3N2、BV流感刺激后γ T細(xì)胞的CD25、CD69以及CD25+CD69+標(biāo)志平均表達(dá)水平分別達(dá)到24.60%、40.11%、22.23%；17.79%、29.81%、15.24%；16.87%、29.84%、15.63%�；罨螃� T細(xì)胞存在一定程度的CD4+CD8+功能型細(xì)胞6.22%、4.14%和3.48%。 3. H1N1、H3N2、BV流感病毒刺激γ T細(xì)胞24小時(shí)候，活化的γ T細(xì)胞對(duì)同一病毒感染的MDCK和MDM細(xì)胞系的殺傷水平分別達(dá)到43.59%、36.76%、21.59%和42.94%、38.15%、23.13%，均表現(xiàn)為較高的殺傷功效。甲型流感病毒活化的γ T細(xì)胞表現(xiàn)出較高的交叉殺傷功效，以H1N1刺激后的γ T細(xì)胞的交叉殺傷作用最強(qiáng)，對(duì)H3N2和BV感染MDM的殺傷作用平均分別達(dá)到36.99%和18.16%；H3N2活化的γ T細(xì)胞地交叉殺傷作用次之，，對(duì)H1N1和BV感染MDM的殺傷作用平均分別達(dá)到29.99%、10.63%。 4.氟伐他汀通過甲羥戊酸途徑抑制流感病毒對(duì)γ T細(xì)胞的活化和功能型γ T細(xì)胞的殺傷作用，表現(xiàn)為經(jīng)氟伐他汀前期處理γ T細(xì)胞，H1N1、H3N2、BV對(duì)γ T細(xì)胞的活化水平下降。其中使各H1N1實(shí)驗(yàn)組γ T細(xì)胞的CD25單陽、CD69單陽以及CD25、CD69雙陽性活化標(biāo)志較刺激前下降56.14%、57.71%和62.12%；H3N2組分別下降40.92%、57.63%和57.55%；BV組分別下降30.88%、39.24%和42.87%。直接殺傷作用H1N1、H3N2、BV各組分別下降42.77%、75.66%和60.63%；H1N1組對(duì)H3N2、BV感染靶細(xì)胞交叉殺傷作用分別下降67.96%和51.28%，H3N2組對(duì)H1N1、BV感染靶細(xì)胞交叉殺傷作用分別下降80.17%和56.52%，BV組對(duì)H1N1、H3N2感染靶細(xì)胞交叉殺傷作用分別下降10.06%和14.48%。 結(jié)論 1.正常人群外周血中T細(xì)胞、γ T細(xì)胞處于未激活狀態(tài)，細(xì)胞主要為CD4CD8雙陰性類別，有部分CD8單陽性細(xì)胞，幾乎沒有CD4陽性細(xì)胞。 2.流感病毒能體外早期（24小時(shí)內(nèi)）快速活化γ T細(xì)胞，且活化能力無型別、亞型間的差異；流感病毒刺激活化后γ T細(xì)胞有趨勢(shì)分化成效應(yīng)性T細(xì)胞，細(xì)胞主要向CD4單陽和CD4CD8雙陽方向發(fā)展。γ T細(xì)胞的分化受型和亞型影響，以H1N1刺激γ T細(xì)胞向CD4CD8雙陽性功能型細(xì)胞分化最為明顯。 3.流感病毒刺激γ T細(xì)胞活化，開啟γ T細(xì)胞特異性殺傷作用和交叉殺傷作用；且已甲型流感（H1N1、H3N2）刺激后表現(xiàn)明顯。 4. γ T細(xì)胞的活化和殺傷作用受甲羥戊酸途徑影響，甲羥戊酸途徑極可能是流感病毒刺激活化γ T細(xì)胞的潛在途徑。
[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.
【學(xué)位授予單位】：中山大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R373.13

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