HSV-1感染对人视网膜色素上皮细胞生长的影响

目的:建立单纯疱疹病毒1型(Herpes simplex virus,HSV-1)感染人视网膜色素上皮细胞(Retinal pigmentepith elial,RPE)的体外模型,观察HSV-1感染对体外培养RPE细胞生长的影响。方法:以感染复数(MOI)为5的HSV-1(F株)感染体外培养的RPE细胞。用相差显微镜观察RPE细胞的形态变化;用MTT法、流式细胞术观察HSV-1感染对RPE细胞增殖、凋亡和细胞周期的影响;用PCR和免疫荧光法验证HSV-1感染RPE细胞是否成功。结果:成功在体外建立了HSV-1感染RPE的细胞模型。HSV-1感染RPE细胞8h后出现病变,细胞形态开始变得细长和皱缩;感染24h后细胞病变更加明显,细胞间间隙增加,甚至可见多核巨细胞;感染48h后所有细胞均变圆,细胞开始出现脱落甚至死亡。MTT法显示HSV-1感染RPE后24h和48h能抑制细胞生长(P<0.01),细胞的抑制率分别为(16.37±3.28)%和(47.91±6.39)%;流式细胞仪检测发现HSV-1感染可影响RPE细胞的细胞周期,但对细胞凋亡无明显影响。结论:HSV-1能感染体外培养的RPE细胞,抑制RPE细胞的增殖,并影响RPE细胞的细胞周期。眼科学报2007;23:212-218.     

环孢霉素A抑制人视网膜色素上皮细胞增生的体外研究

目的 观察环孢霉素A(cyclosporine,CsA)对体外培养人视网膜色素上皮(retinal pigment epithelial,RPE)细胞增生的影响作用.方法 取体外培养人RPE细胞进行分组实验,实验分对照组和实验组:对照组用DMEM培养液继续培养;实验组按所加CsA浓度的不同分为0.20 mg·L-1、0.40 mg·L-1、0.80 mg·L-1、1.60 mg·L-1、3.20 mg·L-1、6.40 mg·L-1.时间效应关系分别于CsA作用24 h、48 h、72 h后测定.对照组和实验组均用MTT比色法测吸光度A值.结果 CsA浓度为0.20 mg·L-1时,作用72 h后差异有统计学意义(P＜0.05);CsA浓度≥0.40 mg·L-1时,作用24 h即差异有统计学意义(P＜0.05).结论 CsA能够抑制体外培养的RPE细胞的生长,这种抑制作用在一定浓度范围内有剂量时间效应.     

去整合素对人眼晶状体上皮细胞的粘附移行作用

目的研究去整合素对体外培养的人眼晶状体上皮细胞生长的抑制作用。方法对先天性白内障患者术中环行撕前囊膜,进行原代培养并传代,取第2代细胞用于实验。抗粘附实验:将悬浮的细胞与不同浓度的去整合素(5μg·L-1、10μg·L-1、20μg·L-1、40μg·L-1、80μg·L-1)在37℃、50mL·L-1CO2条件下一起孵育30min。然后接种于涂有鼠尾胶原的96孔培养板。8h后,用HBSS轻洗各孔2次,MTT法测定粘附细胞的吸光度。抗移行实验:对融合的细胞进行划线,80μg·L-1的去整合素进行干预,24h、48h、72h后,细胞计数法观察裸露区内细胞的移行数量。结果不同浓度的去整合素(20μg·L-1、40μg·L-1、80μg·L-1)对晶状体上皮细胞的粘附均具有抑制作用且呈剂量依赖性。与对照组相比,24h、48h、72h内80μg·L-1的去整合素可显著抑制晶状体上皮细胞的移行。结论去整合素抑制体外培养的人眼晶状体上皮细胞的粘附及移行。     

曲安奈德体外对人视网膜色素上皮细胞功能和结构的影响

目的:探讨曲安奈德(triamcinolone acteonide,TA)对人视网膜色素上皮(retinal pigment epithelium,RPE)细胞功能和活性的影响.方法:通过MTT(四甲基偶氮唑盐比色法),细胞移行实验,细胞黏附实验,RT-PCR检测不同浓度TA(包括含赋形剂的和去除赋形剂的,以及纯赋形剂)在不同时间段对RPE细胞增殖,RPE细胞移行,RPE细胞黏附和线粒体DNA(mitochondrialDNA,mtDNA)损伤的影响.结果:浓度大于0.1g/L含赋形剂和不含赋形剂的TA以及单纯赋形剂对细胞增殖有明显的抑制作用(P＜0.05).细胞移行实验:0.1g/L浓度TA在3个时间点与含血清对照组相比,对100mL/L血清刺激的RPE细胞的移行有显著的抑制作用(P＜0.05).在24,48h与含血清对照组相比各实验组对RPE细胞的黏附有显著的抑制作用(P＜0.05).不同浓度TA(含赋形剂)与RPE细胞作用24h后,结果mtDNA缺失片段不明显,未见明显差异,长片段明显,各组无明显差异.结论:TA对RPE细胞功能(移行能力,黏附能力,增殖能力)有抑制作用,对RPE细胞mtDNA影响不明显.     

实验性视网膜脱离时神经生长因子对视网膜的保护作用

目的　研究神经生长因子 (nervegrowthfactor,NGF)在实验性视网膜脱离 (retinaldetachment,RD)时对视网膜神经细胞的保护作用。方法　 31只Spregue Dawley(SD)大鼠 (6 2只眼 )视网膜下注射透明质酸钠建立RD模型 ,分为 4个组 :NGF实验组 (2 1只眼 )、实验对照组 (2 1只眼 )、病理对照组 (14只眼 )和正常对照组 (6只眼 )。 2 1只SD大鼠右眼玻璃体腔内注射NGF 5 μl(浓度为 1g/L ,1次 / 4d) ,作为NGF实验组 ,左眼注射空白载体作为实验对照组 ,不用药物的 7只SD大鼠 (14只眼 )作为病理对照组。分别在建立RD模型后 12h ,1、2、4、8、16及 32d行光镜和电镜检查 ,进行形态学和细胞数目比较。结果　RD发生后NGF实验组和实验对照组视网膜可见光感受器细胞内、外节缺失、内外核层排列紊乱、神经细胞和神经纤维层水肿变性。实验对照组和病理对照组在镜下表现无明显差异 ,NGF实验组、实验对照组与病理对照组视网膜在RD后 1d镜下即产生明显差异 ,病变程度明显轻于病理对照组和实验对照组 ,以光感受器细胞的内、外节改变最为明显。当RD复位后 ,NGF实验组视网膜组织和细胞结构明显优于病理对照组和实验对照组。细胞计数结果显示 ,随着RD时间延长 ,视网膜细胞核数进行性下降 ,即使视网膜复位 ,各组细胞数仍少于正常对照     

去整合素对人眼品状体上皮细胞的粘附移行作用

目的研究去整合素对体外培养的人眼晶状体上皮细胞生长的抑制作用。方法对先天性白内障患者术中环行撕前囊膜,进行原代培养并传代,取第2代细胞用于实验。抗粘附实验:将悬浮的细胞与不同浓度的去整合素(5μg·L-1、10μg·L-1、20μg·L-1、40μg·L-1、80μg·L-1)在37℃、50mL·L-1CO2条件下一起孵育30min。然后接种于涂有鼠尾胶原的96孔培养板。8h后,用HBSS轻洗各孔2次,MTT法测定粘附细胞的吸光度。抗移行实验:对融合的细胞进行划线,80μg·L-1的去整合素进行干预,24h、48h、72h后,细胞计数法观察裸露区内细胞的移行数量。结果不同浓度的去整合素(20μg·L-1、40μg·L-1、80μg·L-1)对晶状体上皮细胞的粘附均具有抑制作用且呈剂量依赖性。与对照组相比,24h、48h、72h内80μg·L-1的去整合素可显著抑制晶状体上皮细胞的移行。结论去整合素抑制体外培养的人眼晶状体上皮细胞的粘附及移行。     

MTT法检测紫杉醇对兔晶状体上皮细胞增殖的影响

目的　研究紫杉醇 (Taxol)对体外培养的兔晶状体上皮细胞生长的影响。方法　取 2～ 3代对数生长期的兔晶状体上皮细胞 ,于 96孔板中培养 2 4 h后 ,用不同浓度的紫杉醇分别作用 2 4及 72 h,MTT比色测定法观察紫杉醇对兔晶状体上皮细胞增殖的影响。结果　我们发现 ,浓度为 0 .312 5 m g· L- 1的紫杉醇作用于培养的兔晶状体上皮细胞 2 4 h对其生长无明显的抑制 ,而作用 72 h则能抑制其生长。 2 4 h的半效抑制量 (ID50 )为 14 .6 81mg· L- 1 ,72 h的 ID50 为 4 .0 0 7mg· L- 1 ,紫杉醇还能抑制细胞贴壁。结论　紫杉醇对传代培养的兔晶状体上皮细胞增殖有明显的抑制作用 ,紫杉醇对预防后囊膜混浊的发生可能有一定作用     

吡非尼酮对大鼠角膜基质细胞增殖的影响

目的：：探讨吡非尼酮( Pirfenidone,PFD)对体外培养大鼠角膜基质细胞增殖的抑制效果及其对转化生长因子-β1( transforming growth factor-β1,TGF-β1)表达的影响。方法：分离培养大鼠角膜基质细胞,根据PFD用药的不同浓度分为对照组(0mg/mL)、实验组Ⅰ(0.15mg/mL)、实验组Ⅱ(0.3mg/mL)、实验组Ⅲ(1mg/mL),加药48h后应用CCK-8法检测其对角膜基质细胞增殖能力的影响,免疫细胞化学和Western-blot分别检测ki-67和TGF-β1表达的变化。结果：CCK-8结果显示,相比对照组,各实验组对角膜基质细胞的增殖均有明显的抑制作用,且随浓度的增大其抑制作用明显增强(均P<0.05);免疫细胞化学显示PFD能明显降低ki-67阳性指数( P<0.05);Western-blot结果显示,PFD能降低TGF-β1的表达,且呈剂量依赖关系(P<0.05)。结论：PFD对大鼠角膜基质细胞的增殖具有明显抑制作用,抑制机制与下调TGF-β1表达密切相关,在减轻角膜创伤愈合方面具有潜在的运用前景。     

水蛭提取液对人视网膜母细胞瘤细胞的抑制作用

目的：研究水蛭提取液对人视网膜母细胞瘤WERI-RB-1细胞的抑制作用。

方法：采用不同浓度水蛭提取液(0.02、0.04、0.08、0.16U/mL)作用于体外培养的WERI-RB-1细胞0、24、48、72h,经CCK-8法筛选最佳药物干预浓度和时间进行后续实验。将体外培养的WERI-RB-1细胞分为对照组(正常培养基培养)和实验组(含水蛭提取液培养基培养),采用流式细胞仪检测药物对细胞周期和细胞凋亡的影响,Transwell侵袭实验检测药物对细胞侵袭能力的影响。

结果：根据CCK-8法检测结果选择0.04、0.08U/mL水蛭提取液作用48h为最佳干预条件进行实验。水蛭提取液干预的细胞主要阻滞在G2/M期,其中0.04、0.08U/mL实验组处于G2/M期的阳性细胞率分别为(12.59±5.36)%、(14.79±4.12)%,均明显高于对照组\〖(3.00±2.32)%,P<0.01\〗。水蛭提取液可诱导细胞凋亡,其中0.04、0.08U/mL实验组细胞凋亡率分别(37.91±3.44)%、(33.05±2.25)%,均明显高于对照组\〖(4.64±2.56)%,P<0.01\〗。Transwell侵袭实验检测结果显示,实验组Transwell小室下细胞数明显少于对照组,表明水蛭提取液能够抑制细胞侵袭。

结论：水蛭提取液在体外实验中能够抑制人视网膜母细胞瘤细胞的增殖、侵袭,并诱导细胞凋亡。     

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。     

Experimental study on antiviral activity of silicone oil in vitro

OBJECTIVE: To study the antiviral activity of silicone oil against herpes simplex virus-1 (HSV-1) in vitro. METHODS: (1) TCID(50) (Tissue culture infectious dose 50% endpoint) of HSV-1 was titrated. (2) The Hela cells were placed in a 96-well plate. (3) The virus was diluted by 100TCID(50), 50TCID(50), 30TCID(50), 10TCID(50), and 1TCID(50) individually. (4) Each of the five different concentrations of virus was inoculated into 16 wells. Of the 16 wells, 0.1 ml silicone oil was added to eight of them as the experimental group, and the other eight wells were used as controls. (5) Sixteen additional wells were added: silicone oil and maintenance media were added to eight wells, and only maintenance media to the other eight wells. RESULTS: (1) The cytopathic effect (CPE) of wells inoculated with 30TCID(50) combined with silicone oil was significantly less than that of the viral control at 32 hours (P < 0.01), and the same results occurred in group 10TCID(50) combined with silicone oil at 45 hours (P < 0.01) and group 1TCID(50) combined with silicone oil at 51 hours (P < 0.01). (2) In the viral control, the cells in 30TCID(50), 10TCID(50) and 1TCID(50) had pathological changes at 45, 58 and 58 hours respectively. (3) The cells of either the viral control group or group 50TCID(50) and 100TCID(50) had pathological changes at 32 hours (P > 0.05). CONCLUSION: Silicone oil has an antiviral function against HSV-1. The antiviral effect of silicone oil is correlated with concentration of virus.     

Studies of ocular murine cytomegalovirus infection

The pathogenesis of ocular cytomegalovirus (CMV) infection in mice was studied in detail as a model of ocular involvement of human CMV infection. Smith strain of mouse CMV (MCMV) was inoculated into the anterior chamber of the eye and viral antigen was located by immunofluorescence. When salivary gland passaged MCMV (SG-MCMV) was inoculated into the 12- to 18-day-old ICR/Sic mice, it elicited transient uveitis, retinitis, and scleritis during the early phase of infection followed by spread into the lacrimal glands, extraocular muscles and salivary glands. Mouse embryonic fibroblasts (MEF) passaged attenuated MCMV (CC-MCMV) caused mild uveitis with a short duration even inoculated into young mice, and the viral antigen was detected only in salivary glands thereafter. SG-MCMV titer in the eye taken from the young mice decreased from 10(4.5) TCID50/0.1 g at 2 days postinfection (PI) to 10(3.0) TCID50/0.1 g at 21 days PI, whereas in the salivary glands, it became detectable at 5 days PI with a titer of 10(5.0) TCID50/0.1 g, which increased up to 10(8.7) TCID50/0.1 g at 21 days PI. CC-MCMV was detectable only in the young mice salivary glands ranging from 10(4.7) TCID50/0.1 g at 9 days PI to 10(7.3) TCID50/0.1 g at 21 days PI. When the lens capsule had been damaged during the inoculation procedure, the mice developed cataracts with evidence of viral growth in the lens. When SG-MCMV was inoculated onto the cornea, viral antigen was not detected during the period of the present experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility of human corneal endothelial cells to HSV-1 infection

PURPOSE: The purpose of this study was to examine the intrinsic susceptibility of cultured human corneal endothelial cells (HCEC) to herpes simplex virus-1 (HSV-1) infection. We compared HSV-1 adsorption, kinetics of HSV-1 production, and pattern of viral plaque formation in cultured HCEC with those of a cell line used routinely for laboratory HSV propagation (African green monkey kidney fibroblast CV-1 cells). METHODS: Cultured HCEC and CV-1 cells were exposed to the McKrae strain of HSV-1 at 5 and 0.0001 multiplicities of infection (MOI). Using the 50% tissue culture infectious dose (TCID(50)) titration method, viral adsorption (at 5 MOI) and total virus production (at 5 and 0.0001 MOI) were compared to assess both susceptibility to viral attachment and productive viral infection, respectively. Additionally, visual observations were made at 0.0001 MOI using bright-field microscopy and immunofluorescence staining of viral antigens to compare patterns of viral spread in confluent monolayers of both cell types. RESULTS: The percentage of HSV-1 virion particles adsorbed by cultured HCEC and CV-1 cells was similar (35.9% and 33.0%, respectively, p = 0.07, NS), indicating similar susceptibility of the two cell types to initial HSV-1 attachment and adsorption. However, maximum total virus production was more than 3-fold higher for HCEC than for CV-1 cells (p < 0.005), suggesting higher susceptibility of HCEC cells to productive viral infection. Immunofluorescence studies of infected cell monolayers corroborated these quantitative findings, with HCEC monolayers demonstrating more rapid progression of cytopathic effect than CV-1 monolayers. CONCLUSIONS: In comparison to reference CV-1 cells, cultured HCEC show similar susceptibility to HSV-1 adsorption, but higher capacity to support productive HSV-1 infection. Our results suggest that human corneal endothelial cells may be inherently susceptible to HSV-1 infection.     

Therapeutic action of meliacine,a plant-derived antiviral,on HSV-induced ocular disease in mice

Ocular herpes simplex virus type-1 (HSV-1) infections remain an important cause of corneal disease which may result in a loss of vision. Meliacine (MA), an antiviral activity present in crude leaf extracts of Melia azedarach L. that inhibits HSV-1 multiplication in vitro, was studied in a murine herpetic stromal keratitis experimental model. Adult Balb/c mice were inoculated with HSV-1 at their corneas after abrasion. MA was administered topically three times a day for 3 consecutive days, beginning at 24 and 96 hr after infection. Infected animals treated or not with MA were monitored for the development of ocular disease by a binocular microscope for 16 days. MA significantly reduced the incidence and the severity of blepharitis, neovascularization and stromal keratitis with respect to untreated infected mice, regardless the schedule of treatment assayed. Histological examination of corneas from MA-treated animals revealed no tissue damage, whereas corneal samples from untreated infected mice showed inflammation, vascularization and necrosis. In uninfected mice treated with MA, we found no evidence of corneal damage and histopathological studies showed no changes in the corneas of these mice. Treatment with MA at 24 hours post-infection (h.p.i.) reduced viral multiplication in the eye by 1-1.5 orders of magnitude. Studies on latency revealed that MA sligthly affected the establishment of a latent infection. Thus, MA proved to exert an antiviral action on the development of herpetic stromal keratitis when supplied by post-treatment. Unexpectedly, treatment with MA after 96h.p.i prevented ocular disease, suggesting an in vivo immunomodulating activity of MA.     

Subconjunctival antisense oligonucleotides targeting TNF-α influence immunopathology and viral replication in murine HSV-1 retinitis

BACKGROUND: To investigate the role of tumor necrosis factor-alpha (TNF-alpha) in immunopathology and viral replication in the contralateral eye in the von Szily model of herpes simplex virus (HSV)-1 acute retinitis. METHODS: In vivo distribution was analyzed after subconjunctival injection of FITC-labeled antisense oligonucleotides (ASON). After HSV-1 (KOS) was injected in the right anterior chamber (AC) in BALB/c mice, the course of the contralateral retinitis was evaluated. The left eyes were treated with either TNF-alpha ASON, sequence-unspecific control (CON), or buffer. The ocular TNF-alpha content was quantified by ELISA. The delayed-type hypersensitivity (DTH) reaction, uptake of [3H]thymidine from regional lymph nodes (rln)- and spleen cells, serum-neutralizing antibodies, and viral titer in the eyes were evaluated. RESULTS: After subconjunctival injection, FITC-labeled ASON were found in the choroid and retina. In the TNF-alpha ASON-treated eyes, TNF-alpha expression and the incidence and severity of retinitis were reduced on day 8 postinfection (PI) (p < 0.05). On day 10 PI, higher viral titers were only seen in the eyes of the TNF-alpha ASON group (p < 0.05), and retinitis was slightly more severe on day 12 PI. While the HSV-1 specific [3H]thymidine uptake from rln cells was higher in the TNF-alpha ASON mice (p < 0.05), the [3H]thymidine uptake from spleen cells, the DTH response, and the neutralizing-antibody titers did not differ between the groups. CONCLUSIONS: After regional blockade of TNF-alpha in experimental HSV-1 retinitis TNF-alpha seems to possess an antiviral capacity against HSV-1 in the contralateral eye and participates in the immunopathology of HSV-1-induced acute retinitis.     

流式细胞技术测定Ⅰ型单纯疱疹病毒的药物敏感性

目的探讨应用流式细胞技术（FMC）测定Ⅰ型单纯疱疹病毒（HSV-1）对抗病毒药物敏感性的可行性。方法分别应用流式细胞技术和空斑减数试验（PRA）测定对无环鸟苷（ACV）敏感的HSV-1病毒株（SM44）和对ACV耐药的病毒株（ACVr）对常用抗病毒药物ACV、更昔洛韦（GCV）、膦甲酸（PFA）和阿糖腺苷（Ara-A）的敏感性。结果两种检测方法均显示ACVr对ACV和GCV的IC50明显高于SM44（P〈0．05），而对PFA和Ara—A的IC50。两株病毒差异无统计学意义（P〉0．05），FCM法和PRA法的结果具有良好的相关性（r=0．9774，P〈0．01）。结论FCM可以用于抗病毒药物敏感性的测定。     

In vitro sensitivity to antiviral agents of herpes simplex viruses isolated from patients with herpetic keratitis

Thirty-five clinical isolates of herpes simplex virus type 1 (HSV-1) from 34 patients (35 eyes) with herpetic keratitis were examined in vitro for 5-iodo-2'-deoxyuridine (IDU) and acyclovir (ACV) sensitivity. In addition, the effect of clinical treatment with these two drugs in herpetic keratitis was also investigated. The viral effective dose50 (ED50) was defined as the concentration that inhibited the plaque count by 50% compared to the count of the no drug controls. The viral ED50 of IDU ranged from 0.073 to 0.77 micrograms/ml (0.33 +/- 0.16; Mean +/- SD) and that of ACV from 0.0032 to 0.33 micrograms/ml (0.13 +/- 0.11). No virus with markedly diminished sensitivity to IDU and ACV was found. These results suggest that all HSV-1 strains isolated from patients have good sensitivity in vitro to antiviral agents.     

Corneal nerves contain intra-axonal HSV-1 after virus reactivation by epinephrine iontophoresis

Experimental ocular models of herpes simplex virus type 1 (HSV-1) reactivation have been used to monitor viral shedding in the tear film and the appearance of corneal epithelial lesions, but the temporal correlation between reactivation and the presence of viral particles in the corneal nerves has not been made. Two New Zealand white rabbits were inoculated with 20 microliters of HSV-1 McKrae strain (5.0 x 10(6) PFU/ml) in each eye. Beginning on postinfection day 82, ocular iontophoresis (0.8 mAmps for 8 min) of 0.01% epinephrine was done once a day for 3 consecutive days to induce reactivation. Ten limbal nerves from four corneas processed for transmission electron microscopy contained 883 unmyelinated and 40 myelinated axons. Seven nerves were positive for virus. Viral particles were found only in unmyelinated axons, and in low frequency (24/883). Virus was not found in Schwann cells, perineurium, or adjacent stroma nor were virus particles seen exiting axons. No enveloped virions were found. Axons from six nerves of four control corneas from rabbits with latent, but not reactivated, HSV-1 did not contain virus particles. Induction by corneal iontophoresis of epinephrine suggests that HSV-1 is translocated from the ganglion to the cornea through axonal transport mechanisms. For the first time, evidence of anterograde, intra-axonal transport of HSV-1 particles in response to epinephrine reactivation is demonstrated.