单纯疱疹病毒Ⅰ型潜伏感染和复发的分子机制研究进展

单纯疱疹病毒Ⅰ型（HSV－1）引起的单纯疱疹性角膜炎（HSK），是角膜病中的主要致盲原因。因潜伏感染和反复发作而致盲。HSV－1可引起急性感染和潜伏感染。在潜伏期间HSV－1的基因表达和物理状态与在急性感染期间均不相同。在三叉神经节和角膜中发现病毒的基因组和特异的RNA转录，提示角膜中潜伏的HSV－1可能是复发的另一来源。复发是病毒可从港代感染状态激活后再进入溶细胞性感染或基因表达状态。潜伏感染时立刻早期（IE）基因停止转录。有观点认为IE基因转录可启动联级反应导致复发，另有观点认为是一系列的反应着积所致。彻底阐明HSV－1的潜伏和复发机制，是找到防止HSK反复发作而致盲的关键。     

单纯疱疹病毒性角膜炎小鼠模型的建立及鉴定

目的：探讨建立不同感染时期HSK小鼠动物模型,为HSK的深入研究建立基础。方法：Balb/c小鼠125只麻醉后在显微镜下用刀片背面尖端于角膜＂#＂字划痕,其中100只小鼠接种HSV-Ⅰ病毒,另25只小鼠不接种病毒作为正常对照组。术后每天用10g/L荧光素钠染色后裂隙灯显微镜下观察角膜病变发生情况,并取角膜表面泪液进行HEK293T细胞检测以确定裂隙灯显微镜下有无病毒复制。对潜伏感染期小鼠模型采用紫外线B光照射以诱导HSK复发。结果：接种HSV-Ⅰ病毒的小鼠模型眼于接种后3d内全部出现急性上皮性角膜炎表现。经阿昔洛韦滴眼液治疗1wk后角膜炎症消失,但角膜和三叉神经节中PCR检测病毒仍为阳性。潜伏感染期小鼠模型经紫外线B光照射后也都在1wk内复发,并表现为以基质型角膜炎为主要临床表现的角膜病变。结论：采用角膜划痕法对Balb/c小鼠接种HSV-Ⅰ病毒和紫外线B光照射可以成功地制作出原发感染期、潜伏感染期和复发感染期等不同感染时期的HSK模型,而且操作相对简单、方便易行。     

细胞因子在鼠复发性单纯疱疹性角膜基质炎角膜组织中的表达

目的探讨CD4^ T辅助细胞1型(Th1)和T辅助细胞2型(Th2)分泌的细胞因子在鼠复发性单纯疱疹性角膜基质炎(HSK)角膜组织中的表达水平,及其与复发性HSK的关系。方法制备复发性HSK的BALB／c鼠模型,用紫外线B光照射鼠的角膜诱导HSK复发;分别于紫外线照射前,照射后第3、7、10、14、21及28天,取角膜中央直径为2mm的角膜环,用逆转录聚合酶链反应(RT-PCR)检测Th1型细胞因子(IFN-γ、IL-12)和Th2型细胞因子(IL4、IL-10)的表达水平。结果处于病毒潜伏感染期的小鼠经紫外线照射诱导病毒复发后,角膜基质混浊于紫外线照射后的7～14d达到高峰;在此期间,IFN-γ、IL-12、IL-10和IL-4mRNA在角膜组织中均有表达。其中,IFN—γ和IL-10在临床疾病的发生、发展过程中(病毒激活后第3～14天)高表达,在疾病恢复期(14d后)表达减弱,与角膜基质混浊程度密切相关;IL-12是角膜组织内表达最丰富的细胞因子,在病毒激活后的第3天即开始高表达,高表达持续经过整个观察期;IL-4在病毒激活后的第3～14天有明显表达。结论在复发性HSK的角膜损伤早期,Th1型细胞因子和Th2型细胞因子同时表达于角膜组织中,与T细胞的免疫记忆性有关。IL-10的表达趋势平行于IFN-γ的表达,其表达水平与HSK的疾病程度密切相关。复发性HSK无法严格区分是由Th1细胞介导的还是由Th2细胞介导的,角膜的损伤与修复可能依赖于损伤性细胞因子和保护性细胞因子在病毒复发位置上的平衡。     

单纯疱疹病毒性角膜炎的免疫学

单纯疱疹病毒性角膜炎 (herpes simplex keratitis,HSK)是世界范围内角膜盲的最常见原因。单纯疱疹病毒 (herpes simplexvirus,HSV)不仅可直接导致角膜的感染性破坏 ,而且还可通过免疫病理机制诱导某些难以处理的疾病 ,如基质型角膜炎。1　免疫学机制1 .1　动物模型　目前 ,已有数种 HSK的动物模型 ,比较常用的是兔模型和小鼠模型。兔模型比较理想 ,因为兔眼球大小与人类相似易于观察 ;另外 ,存在有自发性病毒释放和疾病复发现象。而小鼠模型则缺乏病毒的自发性释放和疾病的复发 ,但在感染后经过一段时间才发生角膜的炎症反应 ,该特征类…     

单疱病毒角膜炎新分类法评价

目的：评价Holland等提出的单疱病毒角膜炎(HSK）新分类法在指导临床诊治HSK的意义。方法：应用Holland等的新分类法，诊断及治疗门诊各类型单疱病毒角膜炎患者204例，观察其诊断及治疗效果。本组未包括神经营养性角膜病。结果：本组204例HSK中，属于感染性上皮角膜炎86例，其中角膜小泡13例，树枝状溃疡33例，地图状溃疡38例，缘部溃疡2例，全部治愈。属于基质性角膜炎91例，其中坏死性基质角膜炎2例，药物治疗无效，行穿透性角膜移植术。免疫性基质角膜炎89例，全部治愈。属于角膜内皮炎27例，其中中央型13例，周边型8例，弥漫性6例，全部治愈。结论：Holland等的新分类法可指导临床诊治HSK，提高临床诊治质量，改善预后。     

单疱病毒性角膜炎新分类指导临床实践

目的：评价Holland等（1999）提出的单疱病毒性角膜炎（HSK）新分类法在指导临床诊治HSK的意义。方法：应用Holland等的新分类法，诊断及治疗门诊各类型HSK患204例，观察这些患的诊断及治疗效果。本组未包括神经营养性角膜病。结果：本组204例HSK中，属于感染性上皮角膜炎86例，其中角膜小泡13例，树枝状溃疡33例，地图状溃疡38例，缘部溃疡2例，全部治愈。属于基质性角膜炎91例，其中坏死性基质角膜炎2例，药物治疗无效，行穿透性角膜移植术。免疫性基质角膜炎89例，全部治愈。于角膜内皮炎27例，其中中央型13例，周边型8例，弥漫型6例，全部治愈。结论：Holland等的新分类法可指导临床诊治HSK，提高HSK临床诊治质量，改善HSK患预后。     

IL-10对复发性单纯疱疹性角膜基质炎抑制作用的实验研究

目的 研究白细胞介素10（IL-10）在复发性单纯疱疹性角膜基质炎（SK）中的作用。方法 采用复发性单纯疱疹性SK的BALB/c鼠模型，用紫外线B光照射鼠的角膜诱导SK复发。自紫外线照射角膜之前的6h开始，向鼠的角膜内注射重组IL-1010ng，共7次，观察IL-10对复发性S听影响。结果 IL-10减轻了复发性S听角膜混浊程度，减少了角膜内炎性细胞浸润，降低了角膜内IL-1α和IL-6的含量。IL-10没有影响SK的复发率，角膜病毒滴度及迟发型超敏反应。结论 IL-10能抑制角膜细胞产生某些细胞因子：IL-10能抑制复发性单纯疱疹性SK的发展。     

单纯疱疹性角膜炎活动期行穿透性角膜移植术的疗效评价

目的 评价复发型单纯疱疹性角膜炎（herpes simplex keratitis,HSK)基质型活动期行穿透性角移植术的治疗效果。方法 对66例（66只眼）HSK基质型活动期患者行穿透性角膜移植术（penetrating keratoplasty,PKP),并与同期59例（59只眼）HSK基质型稳定期的PKP术后患者的疗效进行对比。结果 两组HSK患者术后角膜植片透明率、原发病复发率及视力间比较差异无显著性（P＞0．05）。结论 HSK基质型活动期行PKP术,只要术前和术中处理得当,不仅可缩短病程和减少并发症,而且还能取得与HSK基质型稳定期行PKP手术同样的疗效。     

单疱病毒性角膜炎大黄素治疗前后补体C3及C反应蛋白的分析

目的：分析中药大黄素对单疱病毒性角膜炎的治疗及免疫调节作用，方法：对62例（86眼）原发感染／复发感染的单疱病毒性角膜炎患者分型，分组，常规用药外施加中药大黄素处理因素进行对照，在二周内观察疗效，时效及补体C3值，C反应蛋白值，结果：原发基质型HSK组的疗效，时效与复发基质型HSK组的时效有统计学意义的改善（团体T检验P＜0．05），原发基质型及复发浅层型HSK组补体C3值实验前后分别上升1．65g/l和1．41g/l；C反应蛋白值实验前后分别上升5．74mg/l和3．39mg/l（团体T－检验P＜0．05），结论：中药大黄素对原发基质型，复发浅层型HSK有改善治疗结果，调节补体C3，促进免疫的作用。     

单疱病毒性角膜炎的新分型及其治疗

目的评价Holland等提出的单疱病毒性角膜炎（HSK）新分类法在诊治HSK时的指导意义及皮质类固醇类药物在治疗HSK时的作用。方法在临床诊治的73例（92眼）HSK患者时应用Holland等提出的新分类法进行分类，再根据不同类型的HSK在抗病毒治疗的基础上灵活应用皮质类固醇类药物进行治疗，观察其临床疗效。结果本组73例（92眼）HSK患者中，感染性角膜上皮炎型42眼，均治愈。基质型角膜炎34眼中，26眼免疫性基质型角膜炎均治愈。3眼坏死性角膜基质炎药物治疗无效行穿透性角膜移植2眼，行深板层角膜移植术1眼。内皮型角膜炎16眼，均治愈。结论Holland等提出的HSK新分类法在临床诊治HSK时具有很好的指导作用，灵活巧妙应用皮质类固醇类药物在治疗HSK时能收到满意的治疗效果。     

Sensory Nerve Retraction and Sympathetic Nerve Innervation Contribute to Immunopathology of Murine Recurrent Herpes Stromal Keratitis

PurposeHerpes stromal keratitis (HSK) represents a spectrum of pathologies which is caused by herpes simplex virus type 1 (HSV-1) infection and is considered a leading cause of infectious blindness. HSV-1 infects corneal sensory nerves and establishes latency in the trigeminal ganglion (TG). Recently, retraction of sensory nerves and replacement with “unsensing” sympathetic nerves was identified as a critical contributor of HSK in a mouse model where corneal pathology is caused by primary infection. This resulted in the loss of blink reflex, corneal desiccation, and exacerbation of inflammation leading to corneal opacity. Despite this, it was unclear whether inflammation associated with viral reactivation was sufficient to initiate this cascade of events.MethodsWe examined viral reactivation and corneal pathology in a mouse model with recurrent HSK by infecting the cornea with HSV-1 (McKrae) and transferring (intravenous [IV]) human sera to establish primary infection without discernible disease and then exposed the cornea to UV-B light to induce viral reactivation.ResultsUV-B light induced viral reactivation from latency in 100% of mice as measured by HSV-1 antigen deposition in the cornea. Further, unlike conventional HSK models, viral reactivation resulted in focal retraction of sensory nerves and corneal opacity. Dependent on CD4⁺ T cells, inflammation foci were innervated by sympathetic nerves.ConclusionsCollectively, our data reveal that sectoral corneal sensory nerve retraction and replacement of sympathetic nerves were involved in the progressive pathology that is dependent on CD4⁺ T cells after viral reactivation from HSV-1 latency in the UV-B induced recurrent HSK mouse model.     

Herpes simplex virus-specific T cells infiltrate the cornea of patients with herpetic stromal keratitis: no evidence for autoreactive T cells

PURPOSE: Herpetic stromal keratitis (HSK) is a T-cell-mediated inflammatory disease initiated by a herpes simplex virus (HSV) infection of the cornea. Recently, studies in the HSK mouse model have shown that the immunopathogenic T cells are directed against the HSV protein UL6 cross-reacting with an unknown corneal autoantigen. Whether this type of autoimmunity plays a role in human HSK was analyzed. METHODS: T-cell lines (TCLs) were generated from corneal buttons of 12 patients with different clinical stages of HSV-induced necrotizing stromal keratitis (n = 9) or immune stromal keratitis (n = 3). The initiating virus was identified by polymerase chain reaction and immunohistology performed on the corneal buttons. Peripheral blood mononuclear cells (PBMCs) were isolated, and B cell lines (BLCLs) were generated by transformation with Epstein-Barr virus. Proliferative responses of these intracorneal TCLs were determined by culturing T cells with autologous BLCLs infected with HSV-1, HSV-2, wild-type vaccinia virus (VV-WT), or VV expressing HSV-1 UL6 (rVV-UL6). Alternatively, T cells were incubated with PBMCs pulsed with human cornea protein extract. RESULTS: Irrespective of clinical diagnosis or treatment, T cells were recovered from the corneal buttons of all the 12 HSK patients. The intracorneal TCLs of 9 of the 12 HSK patients showed HSV-specific T-cell reactivity. In none of the TCLs, T-cell reactivity against HSV-1 UL6 or human corneal antigens was detected. CONCLUSIONS: These data suggest that the potentially immunopathogenic intracorneal T-cell response in HSK patients is directed to the initiating virus and not to a human corneal autoantigen or HSV-1 UL6.     

单纯疱疹病毒性角膜炎动物模型制备

单纯疱疹病毒性角膜炎(herpes simplex keratitis,HSK)是由单纯疱疹病毒Ⅰ型(herpessimplex virus type Ⅰ,HSV-1)感染引起的具有高复发率、高致盲率及难根治性特点的疾病,可导致角膜溶解、新生血管形成及溃疡穿孔,是目前角膜病中最常见的致盲眼病之一.本文参考目前国内外实验研究时使用的各种HSK动物模型建模方法来进行综述,如建立原发感染模型的划痕法、环钻法、外植体培养法以及潜伏感染模型和诱导复发模型的建立方法.力求探索如何建立一种可靠的HSK动物模型,在为研究人类HSK发病机制、药物治疗和对比以及角膜移植等方面均有重要意义.     

Improvement of HSV-1 necrotizing keratitis with amniotic membrane transplantation

PURPOSE: Stromal herpes simplex virus keratitis (HSK) is an immune-mediated disease. Previous studies have indicated that T cells, neutrophils, and macrophages contribute to the tissue damage in HSK. It has been shown that human amniotic membrane promotes epithelial wound healing and has diverse anti-inflammatory effects. In this study, the effect of amniotic membrane transplantation (AMT) on corneal wound healing and on inflammation in mice with necrotizing HSK was examined. METHODS: BALB/c mice were corneally infected with 10(5) plaque-forming units (PFU) of HSV-1 (KOS strain). In 16 mice that exhibited severe ulcerating HSK, the cornea was covered with a preserved human amniotic membrane as a patch. Corneas in 16 infected mice remained uncovered and served as a control. On days 2 and 7 after surgery, the amniotic membrane was removed (eight mice in each group), the HSV-1-infected cornea was evaluated clinically, and the eye was enucleated. Tissue sections were analyzed histologically for epithelialization and cellular infiltration and immunohistochemically with anti-CD3 mAb to T cells, anti-CD11b mAb to both macrophages and neutrophils, or anti-F4/80 mAb to macrophages. RESULTS: Profound regression of corneal inflammation and rapid closure of epithelial defects were observed clinically within 2 days in the amniotic membrane-covered eyes, whereas HSV-1 keratitis and ulceration progressed in all mice in the control group (P < 0.001). Histologically, corneal edema and inflammatory infiltration, and immunohistochemically the number of CD3(+), CD11b(+), and F4/80(+) cells in the cornea were markedly decreased at 2 and 7 days after amniotic membrane application, compared with the uncovered control corneas (P < 0.001). CONCLUSIONS: AMT promotes rapid epithelialization and reduces stromal inflammation and ulceration in HSV-1 keratitis. AMT in mice with HSV necrotizing stromal keratitis appears to be a useful model for investigating the effect and the action mechanism of human amniotic membrane.     

Herpetic stromal keratitis: an immunopathologic disease mediated by CD4+ T lymphocytes.

To investigate the role of T cell subsets in the development of herpetic stromal keratitis (HSK) in a well defined model, we used an adoptive transfer approach in which thymectomized and T cell-depleted mice [T(-)] were reconstituted with different numbers of syngeneic immune T lymphocytes after topical corneal challenge with RE strain of herpes simplex virus-1. In vitro stimulated or unstimulated immune T cells obtained from cervical and retropharyngeal lymph nodes of mice with HSK were used in adoptive transfer experiments. Although T(-) mice developed an initial epithelial inflammation, stromal keratitis did not occur. Reconstitution experiments revealed that mice that received 2 x 10(7) or more unfractionated immune T cells could develop HSK lesions with severity comparable to immuno-competent control mice. In mice receiving CD8(+)-depleted populations, even fewer cells (5 x 10(6)/mouse) were able to induce significant HSK. In contrast, mice that received similar or increased numbers of cells depleted of CD4+ T lymphocytes did not develop HSK. Immune T lymphocytes transferred to mice that were mock infected on the cornea did not develop HSK, indicating that the immunopathogenic cells were virus specific and not merely reacting to autoantigens. Histopathologic examination of the diseased corneas demonstrated that the stromal inflammation in euthymic normal and T(-)-reconstituted mice was characterized by extensive polymorphonuclear leukocyte infiltration. Scattered lymphocytes, and occasional macrophages also were observed. These results provide further evidence that HSK represents an immunopathologic process mediated mainly by CD4+ T cells.