小鼠单纯疱疹病毒性角膜炎中基质金属蛋白酶的表达及活性研究

目的 探讨角膜感染Ⅰ型单纯疱疹病毒(HSV-1)后基质金属蛋白酶(MMPs)及其组织抑制剂(TIMPs)在角膜中的分布及酶活性表达。方法 BALB／c小鼠眼角膜接种HSV-1(KOS株)以诱发单纯疱疹病毒性角膜炎(HSK)。分别收集正常眼球及感染后第2、7、14及28天的感染眼球。应用免疫组织化学法和Western blot方法检测MMP-2、-8、-9及TIMP-1、-2在角膜组织中的表达,并应用酶谱(Zymography)技术检测MMPs的酶活性。结果 感染后第2天,感染眼的MMP-2、-9及TIMP-1、-2表达比未感染眼增加且表达主要位于浅表基质层及上皮下的炎性细胞中。感染后第14和28天可见坏死性角膜炎及角膜溃疡形成,同时角膜基质和浸润的炎性细胞中尤其溃疡处,可见MMP-2、-9及TIMP-1、-2表达显著增加。溃疡区域有大量MMP-8阳性染色的中性粒细胞。角膜感染HSV-1后,明胶酶(MMP-2、-9)活性和胶原酶(MMP-8)活性均增强。结论 HSV-1角膜感染后,由角膜细胞和浸润的炎性细胞分泌产生的MMPs可能对上皮性角膜炎与溃疡形成过程起重要的促进作用。MMPs与TIMPs的相互作用可能对HSK的坏死性病变起重要调节作用。（中华眼科杂志,2004,40：395-399)     

转录因子T-bet在单纯疱疹病毒感染小鼠外周血中的表达

目的研究单纯疱疹病毒Ⅰ型(herpes si mplex virus type1,HSV-1)感染小鼠眼球以后转录因子T-bet在小鼠外周血中的表达,探讨T-bet的表达与单纯疱疹性角膜基质炎之间的关系。方法将106空斑单位(plague forming unit,PFU)·L-1的HSV-1Mckrae毒株接种于BALB/c鼠的角膜上建立单纯疱疹性角膜基质炎(herpetic stromal keratitis,HSK)动物模型,分别于角膜接种病毒后的第1d、3d、7d、10d、14d、21d及28d,用毛细管取小鼠的左眼眼眶静脉窦血1mL,提取淋巴细胞,用半定量逆转录聚合酶链反应(RT-PCR)检测T-bet mRNA的表达水平;在裂隙灯显微镜下观察小鼠角膜的临床变化,组织学检查角膜的病理改变;用ELISA法检测T-bet蛋白在角膜组织中的表达水平。结果BALB/c鼠的角膜接种HSV-1后的1~5d,角膜擦拭液中均检测出HSV-1复制,表明小鼠感染了单纯疱疹病毒;裂隙灯显微镜观察:HSV-1感染鼠的角膜后,小鼠均患了急性角膜上皮炎,并于感染后1周内痊愈。其中81.7%(49/60)的小鼠自感染病毒后第10d起开始出现角膜基质炎改变,表现为灶状角膜基质混浊,局部角膜新生血管化,角膜基质内大量的炎性细胞浸润。角膜基质混浊逐渐进展,在病毒感染后的第14~21d达到高峰。RT-PCR检测的数据显示:未感染HSV-1的对照组小鼠的外周血中不表达T-bet mRNA;HSV-1感染小鼠的早期即可诱导T-bet mRNA在小鼠外周血中的表达,并且表达持续存在;T-bet mRNA表达的高峰时间(10~28d),位于角膜基质炎发生和发展的过程中。ELISA法检测角膜提取物中的T-bet蛋白显示了相似的结果。结论HSV-1上调T-bet mRNA在小鼠外周血中的表达;T-bet的表达与角膜基质炎的发生和发展密切相关。     

HSV-1上调白细胞介素-18 mRNA在小鼠角膜组织中表达的研究

目的研究单纯疱疹病毒Ⅰ型(HSV-1)感染小鼠眼球后,白细胞介素-18(IL-18)在角膜组织中的表达及IL-18的表达与单纯疱疹性角膜基质炎之间的关系。方法用106PFU的HSV-1感染BALB/c鼠的角膜后,在裂隙灯显微镜下观察角膜的临床变化,组织学检查角膜的病理改变;用RT-PCR和ELISA法检测IL-18在角膜组织中的表达水平。结果HSV-1引起的角膜基质炎在病毒感染后的第10d明显可见,在病毒感染后的第14~21d达到高峰。RT-PCR检测的数据显示:HSV-1感染的早期即可诱导IL-18mRNA在角膜组织中的表达,并且表达持续存在;IL-18mRNA的表达高峰时间(3~21d)位于临床疾病出现之前和临床疾病发展的过程中。ELISA检测角膜提取物中的IL-18蛋白显示了相似的结果。结论HSV-1上调IL-18在小鼠角膜组织中的表达;IL-18的表达与角膜基质炎的发生和发展密切相关;IL-18诱导Th1细胞介导的对单纯疱疹病毒特异性的免疫反应,导致单纯疱疹性角膜基质炎。     

可溶性B、T淋巴细胞衰减因子及其配体在单纯疱疹性角膜基质炎小鼠体内的异常表达

目的 研究B、T淋巴细胞衰减因子(B and T lymphocyte attenuator,BTLA)及其配体疱疹病毒侵入介体(herpes virus entry mediator,HVEM)在单纯疱疹性角膜基质炎(herpetic stromal keratitis,HSK)小鼠角膜组织中及外周血CD4+T细胞上的表达水平,探讨共抑制信号BTLA-HVEM是否参与了CD4+T细胞介导的HSK免疫病理反应.方法 将106 PFU的单纯疱疹病毒Ⅰ型(herpes simplex virus type 1,HSV-1)KOS毒株接种于BALB/c鼠的角膜上建立HSK动物模型,分别于角膜接种病毒前(0 d),接种病毒后的第3、7、10、14、21天,用毛细管取小鼠的左眼眼眶静脉窦血1 mL,分离淋巴细胞,行荧光抗体染色,用流式细胞仪检测CD3+ CD4+ BTLA+T细胞和CD3+ CD4+ HVEM+T细胞阳性率;在裂隙灯显微镜下观察小鼠角膜变化;免疫组织化学方法检测BTLA蛋白及HVEM蛋白在角膜组织中的表达.结果 BALB/c鼠的角膜接种HSV-1后的1～5d,角膜擦拭液中均检测出HSV-1复制,表明小鼠感染了单纯疱疹病毒.裂隙灯显微镜观察显示:角膜接种HSV-1后第3天,所有小鼠均患了急性上皮性角膜炎,并于感染后1周内痊愈,自病毒接种后第8天起,小鼠出现角膜基质炎的改变,表现为角膜基质呈灰白色混浊,角膜基质混浊于病毒接种后的第10天达到高峰,持续至第14天后逐渐减轻.流式细胞仪检测显示,小鼠外周血淋巴细胞中CD3+CD4+BTLA+T细胞和CD3+ CD4+ HVEM+T细胞的阳性率,在角膜接种病毒前(0 d)分别为(3.15±0.60)％和(9.84±1.06)％,在角膜接种病毒后第10天(HSK疾病程度最严重时)分别增加到(20.47±3.15)％和(45.18±3.90)％(与0d相比差异均有显著统计学意义,均为P＜0.01).免疫组织化学方法检查HSK小鼠角膜组织中BTLA和HVEM的蛋白表达结果一致:HSK临床表现最严重时,即病毒接种后第10天时,BTLA蛋白和HVEM蛋白在角膜组织中表达最强,主要表达于角膜基质层内浸润的炎性细胞上,角膜上皮层和内皮层也有表达.结论 在HSK小鼠模型中,BTLA及其配体HVEM蛋白在角膜组织中及外周血CD4+T细胞上表达明显增强,共抑制信号BTLA-HVEM参与了CD4+T细胞介导的HSK的免疫病理过程.     

单纯疱疹病毒Ⅰ型在兔角膜内潜伏和活化的实验研究

在健康新西兰兔角膜上建立HSK模型，待其病变稳定期，一组行自体穿透性角膜移植，另一组与未感染HSV-1的兔角膜行交叉穿透移植，拆线后行肾上腺素离子透入拟使潜伏病毒活化。离子透人后的植片抗原检测均为阳性，透射电镜检查显示病毒感染征象，植片角膜基质细胞培养发生HSV—1自发感染。实验结果显示角膜组织是HSV—1的另一个潜伏感染处和复发源地。     

实验性家兔HSV-1角膜炎的扫描电镜改变

本文是一项实验性 HSV-1角膜炎的扫描电镜对照观察。旨在说明在感染的上皮细胞出现众所周知的病毒致细胞病变效应之前,扫描电镜下是否有亚微结构改变,以及细胞破坏在扫描电镜下的形态如何。6只家兔角膜按种 HSV-1,引起角膜炎,6只做对照组。接种后第3天感染的角膜上皮细胞呈现大量直径0.2-2.7μm 的微孔,孔旁堆积有不定形物质。这些细胞表面的微崤或微绒毛未全消失,细胞连接完整。同时,另一些细胞表面出现直径10μm 的小溃疡及2-3μm 的微溃疡。这些细胞的正常表面结构消失,细胞之间出现缝隙。类似的改变也见于感染后5-6天的病损边缘上皮细胞及溃疡底部新露出的上皮细胞。由结果得到如下印象:1.细胞表面的微孔是 HSV-1所致早期亚微改变;2.小及微溃疡也属这种病变,但它们显然是细胞局部破坏的表现,其病变程度似较微孔严重。     

单纯疱疹病毒性角膜炎角膜组织病毒潜伏感染的PCR检测分析

 目的  探讨通过穿透性角膜移植获取的单纯疱疹病毒性角膜炎病变角膜组织中1型单纯疱疹病毒（HSV-1）DNA的表达情况及意义。设计 实验研究。研究对象  2010年5-12月北京同仁医院因病毒性角膜炎角膜白斑行穿透性角膜移植术后角膜标本20例,圆锥角膜、大泡性角膜病变和角膜营养不良等非感染性角膜病变的角膜标本20例。方法  对角膜组织标本中HSV-1 DNA进行聚合酶链反应（PCR）检测。 主要指标  HSV-1 DNA的阳性率。结果  单纯疱疹病毒性角膜炎静止期患者角膜组织中12/20例（60%）检出HSV-1 DNA,非感染性角膜组织中6/20例（30%）检出HSV-1 DNA（χ2=3.64,P=0.057）。结论  单纯疱疹病毒性角膜炎静止期角膜组织多数表达HSV-1 DNA,角膜内潜伏病毒是引起单纯疱疹病毒性角膜炎的可能原因,正常人角膜也可能有HSV-1的DNA存在。（眼科,2013,22：45-48）     

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。     

兔角膜基质细胞培养及对病毒敏感性的实验研究

目的 研究体外培养兔角膜基质细胞对病毒的敏感性。方法 采用微量细胞病变法,观察体外培养的兔角膜基质细胞对单纯疱疾病毒Ⅰ、Ⅱ型(herpes simplex virus Ⅰ、Ⅱ,HSV-Ⅰ、Ⅱ)、腺病毒3型(adenovirus type 3,Ad3)、滤疱性口腔炎病毒(vesicularstomatitis virus,VSV)等4种病毒的敏感性。结果 病毒接种于兔角膜基质细胞48h,HSV-Ⅰ和HSV-Ⅱ效价(TCID_(50))分别为64×10~(-3)、128×10~3,VSV效价(TCID_(50))为10~(-2),Ad3不引起细胞病变。结论 兔角膜基质细胞对HSV-1、HSV-2均敏感,对VSV、Ad3不敏感。     

单纯疱疹病毒Ⅰ型在角膜潜伏感染的研究

眼部感染HSV-1后,在支配该区域的三叉神经节(TG)中建立潜伏感染的理论已被公认,但HSV-1在眼部潜伏感染的确切机理仍不十分清楚。近年众多的研究表明,HSV-1除了在TG潜伏感染外,角膜组织本身也极可能是另一个潜伏感染地。随着组织器官培养和病毒分离技术的提高、DNA探针的应用,为角膜潜伏感染的研究提供了新的、有效的方法,这对全面揭示HSV-1在眼的潜伏感染机制又迈进了一步,将为临床上HSK的治疗提供新的治疗方法和理论依据。     

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.     

OX40-Ig融合蛋白对鼠单纯疱疹性角膜基质炎的抑制作用

目的研究OX40-Ig融合蛋白对鼠单纯疱疹性角膜基质炎(HSK)的免疫抑制作用。方法将1×106PFU的单纯疱疹病毒1型(HSV-1)Mckrae毒株接种于BALB/c鼠的角膜上建立HSK模型;分别于接种病毒的当天、接种后第2、4d将OX40-Ig融合蛋白100μg注射到鼠的腹膜下,观察OX40-Ig融合蛋白对鼠HSK的影响。结果OX40-Ig融合蛋白使鼠外周血中CD4 T细胞减少了78·2%,使鼠HSK发病率由83·3%下降到20·0%。OX40-Ig治疗组的小鼠角膜基质混浊程度较对照组明显减轻,角膜内炎性细胞浸润也明显减少,迟发型超敏反应能力显著下降。结论OX40-Ig融合蛋白能够阻断OX-40/OX-40L协同刺激途径,抑制CD4 T细胞增生,阻止HSK的发病,减轻HSK的严重程度。     

基质金属蛋白酶在实验性单纯疱疹性角膜炎中的分布表达

目的 明确角膜感染Ⅰ型单纯疱疹病毒(HSV—1)后基质金属蛋白酶(MMPs)及其组织抑制剂(TIMPs)在角膜中的分布。方法 HSV—1(KOS株)接种于BALB／c小鼠角膜上。分别收集正常眼球及感染后第2、7、14及28d的感染眼球行石蜡包埋，并应用抗MMP—2、—8、—9及TIMP—1、—2的抗体免疫染色角膜切片。结果 感染后第2d，MMP—2、—9及TIMP—1、—2的表达比未感染眼增加且表达主要位于浅表基质层及上皮下的炎性细胞中。感染后第14d及28d可见坏死性角膜炎及角膜溃疡形成，角膜基质中及浸润的炎性细胞中尤其是溃疡处可见MMP—2、—9及TIMP—1、—2表达显著增加。溃疡区域可见大量MMP—8阳性染色的中性粒细胞。结论 HSV—1角膜感染后由角膜细胞及浸润的炎性细胞分泌产生的MMPs对于上皮性角膜炎及溃疡形成过程可能起重要作用。     

复发性单疱病毒性角膜炎实验模型的研究

目的：建立一种可靠、实用的复发性单疱病毒性角膜炎（HSK）的实验模型。方法：用单纯病毒I型（HSV－1）Mckrae株行NIH鼠的角膜接种,用人的抗HSV－1血清行鼠的腹腔内注射,使病毒在三叉神经节或角膜内建立起潜伏感染。用紫外线B光照射鼠的角膜,诱导HSK复发。观察诱导HSK复发的成功率、复发性HSK的临床特征、组织学特点。结果：紫外线照射诱导鼠HSK复发的成功率为72．5％,复发性HSK主要表现为基质型角膜炎,组织切片见角膜基质层内有大量的淋巴细胞和一些中性粒细胞浸润。     

Activated inflammatory infiltrate in HSV-1-infected corneas without herpes stromal keratitis

PURPOSE: To investigate herpes stromal keratitis (HSK) immunopathology by studying HSV-1-infected corneas that fail to develop HSK. METHODS: Plaque assay quantified HSV-1 in the tear film of infected mice. FACS analysis enumerated corneal leukocytic infiltrate and characterized infiltrate phenotypically after staining for activation and regulatory T cell (Treg) markers and for markers of antigen-presenting cell (APC) maturation. Treg cells were depleted in vivo using anti-CD25 mAb. Luminex analysis quantified the amount of cytokines and chemokines expressed in corneal tissue homogenate. RESULTS: Infected corneas without HSK exhibited a pronounced leukocytic infiltrate containing a significantly higher proportion and nearly identical absolute number of activated CD4+ T cells 15 days after infection when compared with those with HSK. Moreover, the frequency and absolute number of regulatory CD4+ T cells (Tregs) was lower in nondiseased corneas, and Treg depletion did not influence HSK incidence. The frequency of mature, immunogenic DCs and the ratio of mature DCs to CD4+ T cells were nearly identical in corneas with and without HSK. The authors observed a reduced population of neutrophils and reduced expression of neutrophil chemoattractants MIP-1beta and keratinocyte chemoattractant and the neutrophil-attracting cytokine IL-6 in corneas without HSK. CONCLUSIONS: These findings demonstrate that HSV-1-infected corneas can retain clarity in the presence of a substantial secondary leukocytic infiltrate, that activated CD4+ T cells, while necessary, are not sufficient for HSK development, that susceptibility to HSK is not determined by Tregs, and that clinical disease correlates with the accumulation of a critical mass of neutrophils through chemoattraction.     

用原位杂交法检测单纯疱疹病毒Ⅰ型在潜伏感染时的少量潜伏相关转录本表达

目的探讨单纯疱疹病毒Ⅰ型在三叉神经节内潜伏感染时的基因表达。方法以 ＢＡＬＢ／ｃ小鼠制备单纯疮疹性角膜炎,种毒后３６天取三叉神经节和角膜,用免疫组化法检测病毒的抗原表达。制备ｃＲＮＡ探针,用原位杂交法检测 ｍ－ＬＡＴ的表达。结果种毒后３６天,免疫组化结果为阴性,原位杂交结果为阳性。结论 ＨＳＶ－１在三叉神经节内潜伏感染时基因的转录并未静止,有ｍ－ＬＡＴ基因的表达。     

Histological and immunopathological analysis of T-cells mediating murine HSV-1 keratitis

Background: Thymusderived lymphocytes play a critical role in the development of herpes simplex keratitis (HSK). T-cell subsets defined by their expression of various T-cell receptor (TCR) Vß segments were studied following corneal HSV-1 infection (p.i.). Methods: Conjunctiva, corneal limbus and corneal stroma of two inbred BALB/c congenic mouse strains which differ only in the gene products closely linked to the Igh-1 locus on chromosome 12 were analyzed. Results: While C.B-17 mice (Igh-1^b) were resistant to HSK, C.AL-20 mice (Igh-1^d) clinically developed severe necrotizing keratitis by day 11 p.i. The corneal stroma of C.B-17 mice remained clear, while it was increasingly infiltrated by mononuclear cells and neutrophils in C.AL-20 mice by day 11 p.i. In C.B-17 mice, Thy1.2+ cells were found in the conjunctiva between days 2 to 4 p.i., and subsequently decreased. Only a few Thyl.2+ cells were found in the limbus, and no such cells were found in the stroma. In contrast, in C.AL-20 mice the numbers of Thyl.2+ cells (activated CD4+, Vß8+ T cells) profoundly increased in the conjunctiva by day 4 p.i. These cells infiltrated the limbus between days 7 and 11 p.i. and eventually entered the stromal tissue by day 11 p.i. Conclusions: Our data suggest that the HSV-1-induced corneal tissue destruction is mediated by mononuclear cells and neutrophils and that these cells are probably attracted into the cornea by cytokines elaborated by activated CD4+, Vß8+ T cells.Presented as a paper at the ECORA Meeting, 4–7 October 1993, Bonn     

Macrophage-depletion influences the course of murine HSV-1 keratitis

PURPOSE. Herpes simplex virus type 1 infection of the cornea induces an immune-mediated disease termed "herpes stromal keratitis" (HSK) that is a major cause of blindness. In this study we investigated the influence of macrophage depletion by Cl(2) MDP encapsulated in liposomes (Cl(2) MDP-LIP) on the course of HSV-1 keratitis. METHODS. The corneas of BALB/c mice were infected with 10(5) PFU of HSV-1 (KOS strain). Mice groups received sub-conjunctival PBS or Cl( 2) MDP-LIP injections 7 and 2 days prior to infection. The eyes were studied clinically, histologically and immunohistochemically with F4/80 antibody at various time points after treatment. Clearance of the virus from the HSV-infected eyes was measured with a standard plaque assay. RESULTS. After subconjunctival Cl(2) MDP-LIP treatment, the HSV-1-induced epithelial keratitis was more severe (P < 0.05). The virus titers were significantly higher after macrophage depletion (day 7, P < 0.005). Stromal keratitis developed in 78.6% of HSV-1 infected PBS treated control mice ( n = 14) by day 14 after infection. By subconjunctival Cl(2) MDP-LIP treatment (n = 14) the incidence of stromal keratitis was reduced to 42.9%, and the keratitis was less severe (P < 0.05). CONCLUSIONS. The data demonstrate an influence of macrophages on the course of HSV-1 keratitis in mice. Macrophage depletion influence the viral replication in the cornea and the immune-mediated process of HSK.