利用核酸原位杂交技术对HSV－1角膜内潜伏感染的研究

研究单纯疱疹病毒Ｉ型（ｈｅｒｐｅｓｓｉｍｐｌｅｘｖｉｒｕｓｔｙｐｅ１，ＨＳＶ－１）在角膜内的潜伏感染，利用核酸原位杂交技术对１４只新西兰兔双眼角膜基质内注射接种ＨＳＶ－１Ｍｃｋｒａｅ株，２２只限出现典型的原发单疱病毒性角膜炎（ｈｅｒｐｅｓｓｉｍｐｌｅｘｋｅｒａｔｉｔｉｓ，ＨＳＫ）。病毒接种后６０天，取其中４个典型单疱角膜炎原发病变的角膜片交叉移植到４只未接种病毒的兔眼上，术后２周取下被病毒感染的角膜植片，利用ＨＳＶ－１单克隆ＩｇＧ抗体对植片行ＨＳＶ－１抗原检测，利用生物素标记的全长ＨＳＶ－１ＤＮＡ探针进行核酸原位杂交。结果术后４个角膜植片抗原检测均为阴性，而ＨＳＶ－１ＤＮＡ检测均为阳性。提示从分子水平上进一步证明角膜组织极可能是ＨＳＶ－１的潜伏感染地。     

利用核酸原位杂交技术对HSV—1角膜内潜伏感染的研究

研究单纯疱疹病毒Ｉ型（ＨＳＶ－１）在角膜内的潜伏感染，利用核酸原位杂交技术对１４只新西兰兔双眼角膜基质内注射接种ＨＳＶ－１ Ｍｃｋｒａｅ株，２２只眼出现典型的原发单疱病毒性角膜炎（ＨＳＫ）。病毒接种后６０天，取其中４个典型单疱角膜炎原发病变的角膜片交叉移植到４只未接种病毒的兔眼上，术后２周取下被病毒感染的角膜植片，利用ＨＳＶ－１单克隆ＩｇＧ抗体对植片行ＨＳＶ－１抗原检测，利用生物素标记的全长ＨＳＶ     

单纯疱疹病毒Ⅰ型眼部潜伏感染的实验研究

纯系新西兰大白兔经角膜接种ＨＳＶ－ｌｓｔｏｋｅｋ株，成功建立单疱病毒性角膜炎模型。自愈后４５天，以原病变角膜为供体，健康新西兰白兔为受体，行穿通性角膜移植术（ＰＫＰ）同时，取患侧１０份及健侧１０份三叉神经节，应用多聚酶链反应（ＰｏｌｙｍｅｒａｓｅＣｈａｉｎＲｅａｃｔｉｏｎ，ＰＣＲ）检测ＨＳＶ－１ＤＮＡ。术后５０天，取术侧角膜植片１０只及术侧三叉神经节１０份作相同检测。结果表明，患侧三叉神经节１０份中８份ＨＳＶ－１呈阳性，而健侧１０份中３份阳性；角膜植片１０只有７只阳性，术侧三叉神经节未检测到ＨＳＶ－１ＤＮＡ。由此提示，单纯疱疹病毒Ｉ型（ＨＳＶ－１）既可在同侧三叉神经节形成潜伏感染，又可能在对侧三叉神经节形成潜伏，而且角膜极可能为ＨＳＶ－１的另一潜伏部位     

单纯疱疹病毒I型潜伏感染的分子生物学研究

单纯疱疹病毒Ｉ型（ＨＳＶ－１）在角膜发生原发感染后可在三叉神经节（ＴＧ）及角膜组织内发生潜伏感染。分子生物学研究发现潜伏期ＴＧ及角膜组织中都存在ＨＳＶ－１的ＤＮＡ片段及特异性ＲＮＡ的转录，它们与ＨＳＶ－１的潜伏和复发有着密切的关系。但ＨＳＶ－１在ＴＧ与角膜内的潜伏机制有所不同，对这一问题的进一步研究将有助于对ＨＳＶ－１潜伏和复发机理的彻底阐明。     

单纯疱疹病毒Ⅰ型潜伏感染的分子生物学研究

单纯疱疹病毒Ⅰ型（ＨＳＶ－１）在角膜发生原发感染后可在三叉神经节（ＴＧ）及角膜组织内发生潜伏感染。分子生物学研究发现潜伏期ＴＣ及角膜组织中都存在ＨＳＶ－１的ＤＮＡ片段及特异性ＲＮＡ的转录，它们与ＨＳＶ－１的潜伏和复发有着密切的关系。但ＨＳＶ－１在ＴＧ与角膜内的潜伏机制有所不同，对这一问题的进一步研究将有助于对ＨＳＶ－１潜伏和复发机理的彻底阐明。     

原位聚合酶链反应检测角膜组织中HSV—1型DNA

为研究单纯疱疹病毒Ⅰ型（ＨＶＳ－Ⅰ）在角膜内的潜伏感染情况。采用原位聚合酶链反应（ＩＳＰＣＲ）定位检测２１例角膜标本中的ＨＳＶ－Ⅰ，其中７例活动期ＨＳＫ，１３例静止期ＨＳＫ，１例圆锥角膜。结论：在ＨＳＫ活动期及静止期中，角膜各层细胞内均有ＨＳＶ－Ⅰ的存在，极可能整合于细胞核内     

复发性单疱病毒性角膜炎稳定期角膜内潜伏病毒活化的研

应用免疫组织化学染色技术，对２０例稳定了４个月至５年的单疱病毒性角膜炎的每片病变角膜片的一半行单疱病毒Ｉ型抗原检测，结果均为阴性。另一半角膜片经组织活化培养３－４周后再做ＨＳＶ－Ｉ抗原，１８／２０（９０％）阳性。２０例活化后的角膜片与ＲＫ细胞共同培养１周后，有３份ＲＫ细胞出现ＣＰＥ，且ＨＳＶ－Ｉ抗原检测呈阳性。结果表明：ＨＳＫ稳定期的病毒很可能潜伏到角膜组织细胞内，使抗原表达阴性而未被检出，免疫病     

复发性单疱病毒性角膜炎稳定期角膜内潜伏病毒活化的研究

应用免疫组织化学染色技术，对２０例稳定了４个月至５年的单疱病毒性角膜炎（ＨＳＫ）的每片病变角膜片的一半行单疱病毒Ⅰ型（ＨＳＶ－Ⅰ）抗原检测，结果均为阴性。另一半角膜片经组织活化培养３～４周后再做ＨＳＶ－Ⅰ抗原检测，１８／２０（９０％）为阳性。２０例活化后的角膜片与ＲＫ细胞共同培养１周后，有３份ＲＫ细胞出现ＣＰＥ，且ＨＳＶ－Ⅰ抗原检测呈阳性。结果表明：ＨＳＫ稳定期的病毒很可能潜伏到角膜组织细胞内，使抗原表达阴性而未被检出。免疫病理学和病毒学观点提示，ＨＳＫ稳定３个月以上，即可考虑在需要时行穿透性角膜移植术。     

单纯疱疹病毒Ⅰ型潜伏感染复发的分子机制研究进展

单纯疱疹病毒Ⅰ型引起的屯疱疹性角膜炎，是角膜病中的主要致盲原因。因潜伏感染和反复发作作而致盲。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。     

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。     

Persistence of herpes simplex virus DNA in rabbit corneal cells.

Corneal cell cultures were established from the corneas of rabbits killed during a period of latency 118 d after ocular infection with the RE strain of herpes simplex virus (HSV). DNA was isolated from frozen cell pellets of 42 cell cultures that did not develop viral cytopathic effects during 44 d in culture. Using the polymerase chain reaction (PCR) to amplify HSV thymidine kinase (TK) gene sequences, HSV-specific DNA was detected in 15 of 42 culture-negative cell cultures. Subsequent reamplification, using nested primers that were complementary to HSV TK sequences internal to the orginal primers, resulted in eight additional culture-negative samples showing positive hybridization for HSV TK DNA. Twenty three of the 42 virus culture-negative corneal cell cultures tested by PCR were found to contain HSV genetic material. Detailed examination of the clinical histories of the eyes from which the corneal cultures were obtained showed no correlation between increased frequency or severity of epithelial disease, stromal disease, or virus shedding and more frequent isolation of virus or detection of HSV-specific DNA. These studies document that HSV DNA residues in the corneas of HSV-infected rabbits up to 118 d post-infection. About 10% of the eyes contained virus that could be reactivated in culture, whereas an additional 55% of the eyes contained DNA sequences homologous to a portion of the HSV TK gene.     

Peripheral adrenergic stimulation and indomethacin in experimental ocular shedding of HSV

The application of 6-hydroxydopamine to the cornea by iontophoresis, followed by topical epinephrine, effectively induces herpes simplex virus (HSV) shedding from the external eye of latently infected rabbits. In this study the beta adrenergic blocker, Timolol, reduced virus shedding when applied immediately before the epinephrine, but continued administration resulted in increased viral shedding. While indomethacin, a prostaglandin synthesis inhibitor decreased HSV replication in cell culture, it failed to decrease virus shedding when applied topically to the eye in adrenergically stimulated animals. Timolol may act then by its effect on the peripheral cells of the eye rather than by stimulation of virus production in ganglionic neurons. These same animals were subsequently tested for latent infection of the trigeminal and superior cervical ganglia and corneas 14 months after primary infection. Only 2 of 14 animals had virus in the trigeminal ganglia, a finding which suggests that latent virus may be depleted by repeated reactivations. Virus was recovered from corneas of five rabbits by co-cultivation so it is possible that corneal latency occurs in this rabbit model as it does in humans.     

Corneal buttons obtained from patients with HSK harbor high copy numbers of the HSV genome

PURPOSE: To detect herpes simplex virus (HSV) genome in the cornea, we sampled the limbal corneas and scleras of the imported eye bank eyes and recipient's corneal buttons and quantitated HSV genome in them by real-time polymerase chain reaction (PCR). METHODS: Forty-four recipient corneas including 7 corneas with and 37 corneas without a history of herpetic keratitis, 70 eye bank donor limbal corneas, and 35 eye bank donor scleras were obtained. Primers for real-time PCR were synthesized using the HSV-1 and -2 common regions of the viral DNA polymerase. Primers for conventional PCR were designed to detect HSV-1 and -2 and varicella zoster virus (VZV). RESULTS: Significantly higher copy number of HSV DNA was detected in corneas with a history of herpetic keratitis 85.7% (6/7), with an average of 1.6 x 10(4) copies/mg tissue weight than in corneas without a history of herpetic keratitis 10.8% (4/37), with an average of 8.7 copies/mg tissue weight (P < 0.05, Mann-Whitney U test). HSV DNA was detected in 5.7% (4/70) of the eye bank donor corneas, with an average of 4.9 x 10(2) copies/mg tissue weight, and in 8.6% (3/35) of the donor scleras, with an average of 10.6 copies/mg tissue weight. HSV-2 and VZV-DNA were not detected in these samples. CONCLUSIONS: Real-time PCR quantitated HSV genome in the cornea even at a quiescent phase of infection. HSV genome was detected in the corneas and scleras without a past history of herpetic keratitis by this method.     

正常人角膜单疱病毒基因存留的研究

目的研究正常人角膜内单疱病毒（ｈｅｒｐｅｓｓｉｍｐｌｅｘｖｉｒｕｓ，ＨＳＶ）基因存留的可能性。方法用聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）技术对２２例（２２片）正常人角膜、６例（６片）胚胎角膜（阴性对照）及６例单疱病毒性角膜炎（ｈｅｒｐｅｓｓｉｍｐｌｅｘｋｅｒａｔｉｔｉｓ，ＨＳＫ）急性期患者角膜（阳性对照）进行检测。结果２２片正常角膜中２０片存有ＨＳＶＤＮＡ，２片角膜ＨＳＶＤＮＡ阴性，６例胚胎角膜ＰＣＲ为阴性，６例ＨＳＫ急性期患者角膜为阳性。结论正常人角膜中有ＨＳＶＤＮＡ存留，与ＨＳＶ血清抗体阳性结果相符     

The relationship of corneal Langerhans cells to herpes simplex antigens during dendritic keratitis

The corneal migration and topographic distribution of Langerhans cells (LC) in relation to herpes simplex virus antigens was studied during the course of dendritic keratitis in inbred mice. Corneal epithelial sheets from infected mice at selected time points were "double stained" for Ia-positive Langerhans cells and HSV antigens, using a sequential avidin biotin immunoperoxidase and glucose oxidase technique. The amount of HSV antigen was maximum at day 2 paralleling the clinical time course, with most corneal epithelium HSV antigen negative by day 8. LC were seen in peripheral corneas by day 2 and in paracentral and central cornea by day 8, with peak numbers detected between days 8 and 11 post-infection. Although HSV antigens and LC were simultaneously detected within corneal epithelium, LC were not observed in anatomic juxtaposition to HSV antigens, even after reinoculation of infected corneas with HSV on day 14 following the primary infection. These data suggest that local factors in the corneal epithelium other than HSV antigens per se may be chemotactic for LC during the course of dendritic keratitis.