IFN-α调节NK细胞CD100表达在抗HCV感染中的分子机制研究

发布时间：2018-02-09 23:08

本文关键词： 丙型肝炎病毒干扰素-α 自然杀伤细胞 CD100 Plexin-B1/B2　出处：《第四军医大学》2014年博士论文　论文类型：学位论文

【摘要】：丙型肝炎病毒（hepatitis C virus, HCV）感染是全球性严重的健康问题，目前约有1.3~1.7亿人群感染HCV，其中70%~80%的患者发展为慢性。病毒持续感染，部分患者可发展为终末期肝病，如肝硬化和/或肝细胞癌。经过聚乙二醇化干扰素（Pegylated interferon，Peg-IFN）-联合利巴韦林的标准化抗病毒治疗，可使约55%人群获得持续性病毒学应答（sustained virological response, SVR）。因此，充分认识IFN-抗病毒机制尤为重要。IFN-直接抑制病毒复制外，还通过调节机体免疫活性清除HCV感染肝细胞，然而，IFN-抗HCV免疫的潜在分子机制尚需进一步阐明。机体免疫应答在抑制病毒复制和疾病进展中发挥着重要作用。自然杀伤细胞（natural killer cells, NK）作为机体固有免疫系统中的重要组分，通过细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感染过程中，NK细胞参与了抗病毒免疫；多数学者认为，慢性HCV感染中NK细胞处于免疫抑制状态，机制尚未完全清楚。CD100，又称Sema4D，组成性表达于NK细胞和静息T细胞。作为粘附分子，CD100通过与受体结合，增强效应细胞与靶细胞间粘附，对NK细胞功能进行免疫调节。然而，目前尚无关于CD100在慢性HCV感染中研究的实验室证据。本研究选取75例慢性HCV感染者（未治疗30例，应用标准化抗病毒治疗后获得早期病毒学应答者25例，获得SVR者20例）为研究对象。主要采用流式细胞术包膜染色检测外周血NK细胞膜上CD100、CD69和肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis inducing ligand, TRAIL）表达水平；以及采用ELISA检测血清中可溶性CD100（soluble CD100, sCD100）水平。体外观察了HCV感染和IFN-刺激对CD100及受体Plexin-B1/B2表达影响，并通过阻断CD100-Plexin-B1/B2结合，观察NK细胞杀伤活性变化，探讨IFN-介导抗病毒免疫的潜在分子机制及作用模式。主要研究内容和方法： 1.慢性丙型肝炎患者NK细胞CD100表达及意义有研究报道，在免疫缺陷病毒及汉坦病毒感染过程中，CD100参与了机体免疫调节，但目前尚无HCV感染和CD100研究的相关报道。我们主要采用流式细胞术观察了慢性HCV感染者（30例未治疗患者）NK细胞、亚群分布以及NK细胞膜分子CD100，CD69和TRAIL表达水平变化；同时，，采用ELISA法检测了慢性HCV感染者血清中sCD100水平。并采用Spearman相关检验分析了NK细胞膜分子与临床指标，谷丙转氨酶（alanine aminotransferase, ALT）及HCV RNA水平之间的相关性。 2. HCV及IFN-对CD100及受体Plexin-B1/B2表达影响病毒感染可诱导NK细胞活化性受体（NKG2D等）的配体在靶细胞表达，促进NK细胞活化，发挥杀伤功能。众所周知，IFN-是抗HCV感染治疗药物的主要组分，但是其通过调节机体免疫发挥抗病毒作用的分子机制尚不清楚。因此，我们选用K562及Huh7.5细胞做为本部分实验的靶细胞，首先采用qRT-PCR及流式细胞术检测K562及Huh7.5细胞是否表达CD100受体, Plexin-B1/B2。然后，体外采用不同感染复数（1,10,100）的HCV及不同浓度（0.01，0.1,1,10,100,1000ng/ml）的IFN-分别刺激健康人外周血单个核细胞（PBMC）和Huh7.5细胞,在不同时间点观察CD100及其受体Plexin-B1/B2表达水平的变化。 3.探索IFN-调节NK细胞功能的分子作用机制既往研究报道，IFN-通过增强NK细胞功能，发挥抗HCV作用，但是相关分子机制尚不明确。我们首先采用流式细胞术观察了接受Peg-IFN-α联合利巴韦林治疗，获得EVR及SVR者，外周血NK细胞及亚群分布以及CD100，CD69及TRAIL在NK细胞表达变化；同时观察了血清中sCD100水平变化，并分析了CD100，TRAIL与病毒学应答的关系。体外采用K562，Huh7.5及HCV感染的Huh7.5细胞作为靶细胞，与分离的健康人NK细胞共培养12h，在IFN-刺激下，观察NK细胞功能变化；同时采用CD100阻断实验，观察NK细胞毒性及细胞因子IFN-γ分泌变化，探索CD100与其受体结合在调节NK细胞杀伤靶细胞过程中的作用。主要研究结果如下： 1.慢性HCV感染者外周血中NK细胞亚群分布异常，表现为CD56dimNK亚群比率显著下降，“失能”的CD56negNK亚群异常增殖。 2.慢性HCV感染者血清中sCD100水平较健康对照者显著降低；NK细胞CD100、CD69和TRAIL表达轻度异常，但无统计学差异。 3.慢性HCV感染者NK细胞CD100及TRAIL表达分别与谷丙转氨酶水平呈正相关，而与HCV-RNA滴度呈负相关。 4.体外HCV感染及IFN-治疗均显著影响CD100及其受体Plexin-B1/B2表达。 5.慢性HCV感染者经过有效的IFN-抗病毒治疗后获得EVR者，NK细胞CD100、CD69和TRAIL表达显著上调，sCD100血清水平升高；获得SVR的患者，异常分布的NK细胞亚群、血清中sCD100及三种膜分子表达均恢复正常水平。 6. HCV可增强NK细胞毒性，表现为CD107a表达升高，而对IFN-γ分泌无显著影响；对不同靶细胞（K562、Huh7.5及HCV感染的Huh7.5细胞）应答过程中，IFN-显著增强NK细胞杀伤活性，包括毒性及细胞因子分泌功能。 7.阻断CD100与Plexin-B1/B2结合，明显抑制NK细胞功能，表现为CD107a及IFN-γ表达下调。综上所述，我们发现HCV感染通过下调NK细胞CD100表达，降低效应细胞与靶细胞间接触，抑制对靶细胞杀伤和病毒清除。根据慢性HCV感染者CD100与ALT、HCV-RNA水平之间相关性，推测CD100可能参与了免疫介导的肝脏炎症发生和病毒控制。在NK细胞对靶细胞免疫应答中，IFN-治疗通过上调CD100、TRAIL、CD107a和IFN-γ等分子表达，促使NK细胞活化，增强了对病毒感染肝细胞的杀伤，有助于清除HCV。探讨其机制，发现CD100与受体Plexin-B1/B2结合在调节NK细胞功能中发挥了关键作用，为研制抗HCV感染新的药物靶点提供了实验和理论依据。
[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.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.63

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