本文關(guān)鍵詞： 丙型肝炎病毒 干擾素-α 自然殺傷細胞 CD100 Plexin-B1/B2　出處：《第四軍醫(yī)大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：丙型肝炎病毒（hepatitis C virus, HCV）感染是全球性嚴重的健康問題，目前約有1.3~1.7億人群感染HCV，其中70%~80%的患者發(fā)展為慢性。病毒持續(xù)感染，部分患者可發(fā)展為終末期肝病，如肝硬化和/或肝細胞癌。經(jīng)過聚乙二醇化干擾素（Pegylated interferon，Peg-IFN）-聯(lián)合利巴韋林的標準化抗病毒治療，可使約55%人群獲得持續(xù)性病毒學(xué)應(yīng)答（sustained virological response, SVR）。因此，充分認識IFN-抗病毒機制尤為重要。IFN-直接抑制病毒復(fù)制外，還通過調(diào)節(jié)機體免疫活性清除HCV感染肝細胞，然而，IFN-抗HCV免疫的潛在分子機制尚需進一步闡明。 機體免疫應(yīng)答在抑制病毒復(fù)制和疾病進展中發(fā)揮著重要作用。自然殺傷細胞（natural killer cells, NK）作為機體固有免疫系統(tǒng)中的重要組分，通過細胞毒性和分泌抗病毒因子如，IFN-γ等，在早期控制病毒感染。在急慢性HCV感染過程中，NK細胞參與了抗病毒免疫；多數(shù)學(xué)者認為，慢性HCV感染中NK細胞處于免疫抑制狀態(tài)，機制尚未完全清楚。CD100，又稱Sema4D，組成性表達于NK細胞和靜息T細胞。作為粘附分子，CD100通過與受體結(jié)合，增強效應(yīng)細胞與靶細胞間粘附，對NK細胞功能進行免疫調(diào)節(jié)。然而，目前尚無關(guān)于CD100在慢性HCV感染中研究的實驗室證據(jù)。 本研究選取75例慢性HCV感染者（未治療30例，應(yīng)用標準化抗病毒治療后獲得早期病毒學(xué)應(yīng)答者25例，獲得SVR者20例）為研究對象。主要采用流式細胞術(shù)包膜染色檢測外周血NK細胞膜上CD100、CD69和腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體（tumor necrosis factor-related apoptosis inducing ligand, TRAIL）表達水平；以及采用ELISA檢測血清中可溶性CD100（soluble CD100, sCD100）水平。體外觀察了HCV感染和IFN-刺激對CD100及受體Plexin-B1/B2表達影響，并通過阻斷CD100-Plexin-B1/B2結(jié)合，觀察NK細胞殺傷活性變化，探討IFN-介導(dǎo)抗病毒免疫的潛在分子機制及作用模式。 主要研究內(nèi)容和方法： 1.慢性丙型肝炎患者NK細胞CD100表達及意義 有研究報道，在免疫缺陷病毒及漢坦病毒感染過程中，CD100參與了機體免疫調(diào)節(jié)，但目前尚無HCV感染和CD100研究的相關(guān)報道。我們主要采用流式細胞術(shù)觀察了慢性HCV感染者（30例未治療患者）NK細胞、亞群分布以及NK細胞膜分子CD100，CD69和TRAIL表達水平變化；同時，，采用ELISA法檢測了慢性HCV感染者血清中sCD100水平。并采用Spearman相關(guān)檢驗分析了NK細胞膜分子與臨床指標，谷丙轉(zhuǎn)氨酶（alanine aminotransferase, ALT）及HCV RNA水平之間的相關(guān)性。 2. HCV及IFN-對CD100及受體Plexin-B1/B2表達影響 病毒感染可誘導(dǎo)NK細胞活化性受體（NKG2D等）的配體在靶細胞表達，促進NK細胞活化，發(fā)揮殺傷功能。眾所周知，IFN-是抗HCV感染治療藥物的主要組分，但是其通過調(diào)節(jié)機體免疫發(fā)揮抗病毒作用的分子機制尚不清楚。因此，我們選用K562及Huh7.5細胞做為本部分實驗的靶細胞，首先采用qRT-PCR及流式細胞術(shù)檢測K562及Huh7.5細胞是否表達CD100受體, Plexin-B1/B2。然后，體外采用不同感染復(fù)數(shù)（1,10,100）的HCV及不同濃度（0.01，0.1,1,10,100,1000ng/ml）的IFN-分別刺激健康人外周血單個核細胞（PBMC）和Huh7.5細胞,在不同時間點觀察CD100及其受體Plexin-B1/B2表達水平的變化。 3.探索IFN-調(diào)節(jié)NK細胞功能的分子作用機制 既往研究報道，IFN-通過增強NK細胞功能，發(fā)揮抗HCV作用，但是相關(guān)分子機制尚不明確。我們首先采用流式細胞術(shù)觀察了接受Peg-IFN-α聯(lián)合利巴韋林治療，獲得EVR及SVR者，外周血NK細胞及亞群分布以及CD100，CD69及TRAIL在NK細胞表達變化；同時觀察了血清中sCD100水平變化，并分析了CD100，TRAIL與病毒學(xué)應(yīng)答的關(guān)系。體外采用K562，Huh7.5及HCV感染的Huh7.5細胞作為靶細胞，與分離的健康人NK細胞共培養(yǎng)12h，在IFN-刺激下，觀察NK細胞功能變化；同時采用CD100阻斷實驗，觀察NK細胞毒性及細胞因子IFN-γ分泌變化，探索CD100與其受體結(jié)合在調(diào)節(jié)NK細胞殺傷靶細胞過程中的作用。 主要研究結(jié)果如下： 1.慢性HCV感染者外周血中NK細胞亞群分布異常，表現(xiàn)為CD56dimNK亞群比率顯著下降，“失能”的CD56negNK亞群異常增殖。 2.慢性HCV感染者血清中sCD100水平較健康對照者顯著降低；NK細胞CD100、CD69和TRAIL表達輕度異常，但無統(tǒng)計學(xué)差異。 3.慢性HCV感染者NK細胞CD100及TRAIL表達分別與谷丙轉(zhuǎn)氨酶水平呈正相關(guān)，而與HCV-RNA滴度呈負相關(guān)。 4.體外HCV感染及IFN-治療均顯著影響CD100及其受體Plexin-B1/B2表達。 5.慢性HCV感染者經(jīng)過有效的IFN-抗病毒治療后獲得EVR者，NK細胞CD100、CD69和TRAIL表達顯著上調(diào)，sCD100血清水平升高；獲得SVR的患者，異常分布的NK細胞亞群、血清中sCD100及三種膜分子表達均恢復(fù)正常水平。 6. HCV可增強NK細胞毒性，表現(xiàn)為CD107a表達升高，而對IFN-γ分泌無顯著影響；對不同靶細胞（K562、Huh7.5及HCV感染的Huh7.5細胞）應(yīng)答過程中，IFN-顯著增強NK細胞殺傷活性，包括毒性及細胞因子分泌功能。 7.阻斷CD100與Plexin-B1/B2結(jié)合，明顯抑制NK細胞功能，表現(xiàn)為CD107a及IFN-γ表達下調(diào)。 綜上所述，我們發(fā)現(xiàn)HCV感染通過下調(diào)NK細胞CD100表達，降低效應(yīng)細胞與靶細胞間接觸，抑制對靶細胞殺傷和病毒清除。根據(jù)慢性HCV感染者CD100與ALT、HCV-RNA水平之間相關(guān)性，推測CD100可能參與了免疫介導(dǎo)的肝臟炎癥發(fā)生和病毒控制。在NK細胞對靶細胞免疫應(yīng)答中，IFN-治療通過上調(diào)CD100、TRAIL、CD107a和IFN-γ等分子表達，促使NK細胞活化，增強了對病毒感染肝細胞的殺傷，有助于清除HCV。探討其機制，發(fā)現(xiàn)CD100與受體Plexin-B1/B2結(jié)合在調(diào)節(jié)NK細胞功能中發(fā)揮了關(guān)鍵作用，為研制抗HCV感染新的藥物靶點提供了實驗和理論依據(jù)。
[Abstract]:Hepatitis C virus (hepatitis C, virus, HCV) infection is a serious global health problem, there are about HCV million people infected with 1.3~1.7, of which 70%~80% of the patients. The development of chronic persistent viral infection, some patients may develop end-stage liver disease such as cirrhosis and / or hepatocellular carcinoma. After pegylated interferon (Pegylated interferon, Peg-IFN) - standard antiviral therapy combined with Leigh Bhave Lin, can make about 55% people get sustained virologic response (sustained virological, response, SVR). Therefore, to fully understand the mechanism of antiviral IFN- particularly important.IFN- direct inhibition of virus replication, but also through regulating the immune activity of scavenging HCV infected liver cells, however, the molecular mechanisms underlying IFN- immunization against HCV still need to be further clarified.
Immune response plays an important role in the progress of inhibition of virus replication and disease. Natural killer cells (natural killer cells, NK) as an important group in the innate immune system, through cytotoxicity and secretion of antiviral cytokines such as IFN-, gamma, in the early control of virus infection. In acute and chronic HCV infection in the process. NK cells are involved in antiviral immunity; the majority of scholars believe that, in patients with chronic HCV infection of NK cells in immune suppression, the mechanism has not yet entirely clear.CD100, also called Sema4D, is constitutively expressed on NK cells and resting T cells. As adhesion molecules, with the receptor binding CD100, enhancement effect cells and target cells adhesion on. The function of NK cells in immune regulation. However, there is no laboratory evidence on CD100 in patients with chronic HCV infection.
This study selected 75 patients with chronic HCV infection (no treatment in 30 cases, application of standardized antiviral treatment after 25 cases of early virologic response for SVR in 20 cases) as the research object. Using flow cytometry staining envelope detection of peripheral blood NK cell membrane CD100, CD69 and tumor necrosis factor related apoptosis (tumor necrosis factor-related apoptosis ligand induced inducing, ligand, TRAIL) expression level; and the detection of serum soluble CD100 ELISA (soluble CD100 sCD100). Observe the effects of HCV infection and IFN- expression of CD100 and Plexin-B1/B2 affected by the body, and through the combination of block CD100-Plexin-B1/B2, observe the killing activity of NK cells to explore the potential molecular changes. The mechanism and mode of action of IFN- mediated antiviral immunity.
Main research contents and methods:
Expression and significance of NK cell CD100 in 1. patients with chronic hepatitis C
Studies have reported that the human immunodeficiency virus and hantavirus infection process, CD100 is involved in immune regulation, but there is no reports of HCV infection and CD100 related research at present. We mainly use the observation of chronic HCV infection by flow cytometry. (30 cases of untreated patients) NK cell subsets and NK cell membrane molecule CD100, the expression of CD69 and TRAIL; at the same time, the detection of sCD100 levels in sera of patients with chronic HCV infection by ELISA method. And the analysis of the NK cell membrane molecules and clinical indicators using Spearman correlation test, ALT (alanine aminotransferase, ALT) and the relationship between the HCV level of RNA.
Effects of 2. HCV and IFN- on the expression of CD100 and receptor Plexin-B1/B2
Virus infection can induce the activation of NK cell receptor (NKG2D) ligand expression in target cells, promote the activation of NK cells, play killer function. As everyone knows, IFN- is the main group of anti HCV infection therapy, but the molecular mechanism through regulating the immune function of anti-virus is not clear. Therefore, we choose K562 and Huh7.5 cells as target cells in this part of the experiment, firstly using qRT-PCR and flow cytometry was used to detect K562 and Huh7.5 expression of CD100 receptor Plexin-B1/B2. in vitro, then, using different multiplicities of infection (1,10100) and different concentrations of HCV (0.01,0.1,1,101001000ng/ml) IFN- were used to stimulate healthy human peripheral blood mononuclear cells (PBMC) and Huh7.5 cells, the effects of CD100 and its receptor Plexin-B1/B2 expression at different time points.
3. explore the molecular mechanism of IFN- regulating the function of NK cells
Previous studies reported that IFN- enhances NK cell function, play the role of anti HCV, but the molecular mechanism is not clear. We first observed by receiving Peg-IFN- alpha combined with Leigh Bhave Lin in the treatment of flow cytometry, EVR and SVR, peripheral blood NK cells and subsets of CD100 and expression of CD69 and TRAIL. In the NK cells; and to observe the changes of serum levels of sCD100, and analyzes the relationship between CD100, TRAIL and virology. The in vitro response by K562, Huh7.5 and HCV infected Huh7.5 cells as target cells, 12h co cultured with NK cells from healthy people, under the stimulation of IFN-, observe the changes of NK cells and the function; CD100 blocking experiments, observation of NK cell cytotoxicity and cytokine IFN- secretion changes, to explore the binding of CD100 and its receptors in the regulation of NK cells to kill target cells in the process.
The main results are as follows:
1. the distribution of NK cell subsets in peripheral blood was abnormal in the patients with chronic HCV infection, which showed a significant decrease in the CD56dimNK subgroup ratio and the abnormal proliferation of the "dysfunctional" CD56negNK subgroup.
2. the level of serum sCD100 in patients with chronic HCV infection was significantly lower than that of the healthy controls, and the expression of CD100, CD69 and TRAIL in NK cells was slightly abnormal, but there was no statistical difference.
3. the expression of CD100 and TRAIL in NK cells in patients with chronic HCV infection was positively correlated with the level of alanine transaminase, but negatively correlated with the titer of HCV-RNA.
4. in vitro HCV infection and IFN- treatment all significantly affect the expression of CD100 and its receptor Plexin-B1/B2.
5. patients with chronic HCV infection after IFN- effective antiviral therapy after EVR, CD100 CD69 and NK cells, the expression of TRAIL was significantly up-regulated, elevated levels of serum sCD100; SVR patients with abnormal distribution of NK cell subsets, the expression of sCD100 in serum and three kinds of membrane molecules were restored to the normal level.
6. HCV could enhance the cytotoxicity of NK, showing that the expression of CD107a increased, but it had no significant effect on IFN- gamma secretion. During the response of different target cells (K562, Huh7.5 and HCV infected Huh7.5 cells), IFN- significantly enhanced the cytotoxicity of NK cells, including toxicity and cytokine secretory function.
7. blocking the combination of CD100 and Plexin-B1/B2 significantly inhibited the function of NK cells, showing downregulation of CD107a and IFN- gamma expression.
In summary, we found that HCV infection through down-regulation of NK expression of CD100, reduce the effect of contact between cells and target cells, inhibition of target cell killing and viral clearance. According to the CD100 and ALT of chronic HCV infection, correlation between HCV-RNA levels, speculated that CD100 may be involved in immune mediated liver inflammation occurred in NK cells and virus control. The target cell immune response, IFN- treatment through upregulation of CD100, TRAIL, CD107a and IFN- molecular expression of gamma, promote the activation of NK cells, enhance the liver cell killing virus infection, contribute to the removal of HCV. and explore its mechanism, found that CD100 and Plexin-B1/B2 binding receptors play a key role in the regulation of NK cell function and provide experimental and theoretical basis for the development of anti HCV infection of new drug targets.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R512.63

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相關(guān)期刊論文 前1條

1 何瑜;范超;賈戰(zhàn)生;;CD100及其受體在免疫系統(tǒng)中的調(diào)節(jié)作用[J];中國免疫學(xué)雜志;2013年12期

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