增生性玻璃体视网膜病变增殖膜的免疫组织化学及细胞凋亡研究

目的　检测增生性玻璃体视网膜病变 (PVR)增殖膜标本中细胞Fas、Fas配体 (Fasligand ,FasL)的表达和凋亡 ,并探讨Fas/FasL与凋亡的关系。方法　通过免疫组织化学的方法检测 12例增殖膜标本Fas和FasL的表达 ,TUNEL技术检测凋亡细胞。结果　PVR增殖膜标本中Fas和FasL阳性细胞百分率分别为 60 6%和 43 75 % ,偶见两者同时表达的细胞。 5例前膜和 2例下膜标本可见TUNEL染色阳性细胞核。凋亡与Fas、FasL相关系数均为 0 77(t =3 82 ,P <0 0 5 ) ,PVR病程与凋亡的r值为 0 74(t =3 11,P <0 0 1)。结论　Fas、FasL高表达与凋亡的正相关性提示 ,通过Fas/FasL系统诱导增殖细胞及炎症细胞凋亡 ,可能成为临床防治PVR的新策略     

Bak和bcl-xl在增生性玻璃体视网膜病变玻璃体内细胞和视网膜前膜中的表达

目的　观察增生性玻璃体视网膜病变 (PVR)玻璃体内和视网膜前膜 (ERM)中细胞凋亡、bak和bcl xl基因表达、以及细胞凋亡和基因表达之间的关系。方法　对 12例PVRB级玻璃体切割液离心收集物 ,18例C级以上PVR的ERM进行研究 ,末端标记 (TUNEL)染色法检测凋亡细胞 ,免疫组织化学染色法检测bak和bcl xl基因的表达。结果　B级玻璃体切割液标本中bak和bcl xl阳性细胞百分数分别为 73 0 %和 65 1% ,TUNEL阳性细胞百分率平均为 2 7 2 %。C级以上PVR的ERM标本中bak和bcl xl的阳性细胞比例分别为 61 4%和 77 0 % ,TUNEL阳性细胞百分率平均为 18 6%。PVRB级的凋亡细胞百分数及bak的表达与C级以上标本之间相差明显 (P <0 0 5 ,P <0 0 1) ,而bcl xl的表达在两者间没有差别 (P >0 0 5 )。凋亡细胞bak表达与凋亡细胞百分数相关系数r值为 0 86(P <0 0 1) ,与bcl xl的r值为 0 3 2 (P >0 0 5 )。结论　PVR眼增殖细胞中可见bak和bcl xl的表达和细胞凋亡的存在。bak的高表达可能与PVR中增生细胞凋亡有关。     

大鼠角膜碱烧伤后视网膜细胞凋亡的研究

目的研究严重眼前段碱烧伤后视网膜组织内的细胞凋亡情况,以及Fas抗原(Fas)和Fas配体(Fas ligand,FasL)表达与细胞凋亡的关系。方法制作大鼠角膜严重碱烧伤模型。在烧伤的不同阶段,应用TUNEL法检测凋亡细胞、免疫组织化学法检测Fas、FasL在视网膜组织中的表达和变化,并与正常大鼠相对照。结果正常视网膜中基本未见TUNEL阳性细胞,碱烧伤3d后在视网膜神经节细胞、内核层细胞和内层血管内皮细胞中均可见不同程度的TUNEL阳性细胞。正常视网膜未见Fas表达,实验各组中均可见不同程度的Fas表达;正常视网膜FasL弱表达,实验各组FasL表达显著增强,与正常对照组比较差异有显著性(P<0.01)。Fas和FasL表达与凋亡之间具有较好的相关性,相关系数分别为0.899(P<0.01)和0.504(P<0.01)。结论严重眼前段碱烧伤后视网膜组织细胞存在异常凋亡,凋亡可能与Fas/FasL系统有关。     

增生性玻璃体视网膜病变玻璃体切割物的细胞凋亡观察

目的观察增生性玻璃体视网膜病变玻璃体切割物的细胞凋亡情况 。方法收集60例不同分级的增生性玻璃体视网膜病变患者的玻璃体切割物,核苷酸末端转移酶介导dUTP缺口翻译法(TdT-mediated dUTP nick end label ling method,TUNEL法)标记凋亡细胞,光镜观察。结果60例患者的玻璃体切割物均有细胞凋亡的特征性改变,随着病变程度的加重,非色素细胞凋亡总数逐渐减少,并出现色素细胞凋亡。结论增生性玻璃体视网膜病变存在不同类型细胞的凋亡,细胞凋亡是调控其病变程度的重要机制之一。(中华眼底病杂志,1999,15:81-83)     

增生性玻璃体视网膜病变眼玻璃体中差异蛋白质组的鉴定

背景增生性玻璃体视网膜病变（PVR）是导致视网膜脱离手术失败的最主要原因之一。蛋白质组学是研究人体组织中差异蛋白的重要方法。目的应用双向凝胶电泳（2-DE）联合质谱分析法对原发性视网膜脱离伴PVR患者进行差异蛋白质组分析,筛选出PVR眼特异的蛋白质表达。方法经玻璃体切割术收集PVRB级（轻度PVR）,PVRC、D级（重度PVR）患者各8例8眼的玻璃体标本进行（2-DE）联合质谱检测分析,正常供体8眼作为对照。抽取的玻璃体标本再水化液重溶后,加至固相pH梯度胶条（18CIn,pH3—10）水化,等电聚焦,平衡后进行聚丙烯酰胺凝胶电泳,银染。对差异明显的蛋白质点（至少2倍以上的吸光度）进行胶内酶解,质谱鉴定。结果供体眼、轻度PVR眼和严重PVR眼经2-DE图谱分别检测到玻璃体标本中47、184和336个蛋白质点,PVR增加的蛋白质点的pH等电点以5～8居多,轻度PVR和重度PVR分别为104（56．5％）和230点（68．5％）,在13个明显差异点中鉴定到7种蛋白质。其中,烯醇酶为供体眼所特有,甲状腺激素结合蛋白单体和视黄醇结合蛋白（RBP）单链为重度PVR玻璃体中特异表达的蛋白质,前列腺素D合成酶和RBP3前体在供体眼和PVR眼玻璃体均能检测到,且在轻度PVR眼中表达量较高,严重PVR眼下调。玻璃体中血清白蛋白和转铁蛋白随PVR严重程度的增加而明显上调。结论PVR发病过程中,原有的玻璃体蛋白质发生降解,部分血清中的蛋白质明显k调,表明PVR眼有血一视网膜屏障的破坏。     

增殖性玻璃体视网膜病变细胞凋亡的信号传导途径研究

目的 :探讨增殖性玻璃体视网膜疾病细胞凋亡的信号传导途径 ,以寻求新的药物治疗途径。方法 :2 3例增殖性玻璃体视网膜病变 (PVR) ,增殖性糖尿病性视网膜病变 (PDR) ,黄斑裂孔 (MH)及黄斑前膜 (MP)的视网膜前膜 (epiretinalmem brane,ERM )由玻璃体切割术中取得。细胞凋亡的情况由terminaldeoxynucleotidyltransfrase dUTP nickendlabeling(TUNEL法 )进行评估。Caspase 3及PARP的表达由特异性抗体抗活性Caspase 3和抗P 85片段的PARP检测。Cytokeratin与抗活性的Caspase 3双染色法进行凋亡细胞类型的鉴别。结果 :大多数发生凋亡的细胞为RPE细胞 ,而凋亡细胞与抗活性Caspase 3和抗P 85片段的PARP表达增加相关。细胞凋亡的数目与发生慢性视网膜脱离 (>2个月 )的病例有关 ,但凋亡系数 (apopto sisindex ,AI)在两组间无显著性差异 (1 4 4 2 9vs 3 2 2 86 ,P =0 1877)。PVR ,PDR ,MP各组的凋亡系数分别为 2 32 5 % ,3 4 2 % ,5 5 % ,P值分别为PPVR&PDR>0 1(0 16 85 ) ,PPDR&MP>0 1(0 5 380 ) ,PPVR&MP>0 1(0 8333)。结论 :此项研究发现细胞凋亡在PVR、PDR、MH及MP发病中的重要调节作用。诱导Caspase 3活性表达可作为一种治疗增殖性视网膜疾病的新的尝试     

增生性玻璃体视网膜病变增殖膜的免疫组织化学研究

增生性玻璃体视网膜病变 (PVR)是孔源性视网膜脱离(rhegmatogenousretinaldetachment,RRD)手术失败的主要原因。增殖膜的形成、收缩 ,并牵拉视网膜导致视网膜脱离是主要病理过程 ,在以往的研究中我们已经发现PVR患者玻璃体切割液中存在细胞表型的变化[1] ,我们通过对 12例PVR患     

增生性玻璃体视网膜病变基质金属蛋白酶的定量研究

目的:研究增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)玻璃体中基质金属蛋白酶(matrix metalloproteinases,MMPs)的表达,探讨MMPs在PVR病理过程中的作用。方法:PVR患者采用标准三切口巩膜扁平部玻璃体切割术(pars plana vitrectomy,PPV),取未稀释的玻璃体21只眼,PPV术后复发的玻璃体腔液20只眼,意外死亡的正常人玻璃体10只眼,采用明胶酶谱分析法定量分析MMP-2和MMP-9活性水平。结果:PVR玻璃体有MMP-2活性水平增高,与正常玻璃体比较差异有显著性意义(P<0.05)。21眼PVR玻璃体中13只眼有MMP-9活性水平增高,平均(171.52±13.17)扫描单位。20眼PPV术后PVR复发的玻璃体腔液19只眼有MMP-9活性水平增高,平均(156.01±37.21)扫描单位。正常人玻璃体无MMP-9的表达。结论:PVR玻璃体有MMP-2和MMP-9活性水平增高,MMP-9活性水平增高可能与术后PVR复发有关。眼科学报2003;19:130-132。     

α-硫辛酸治疗大鼠视网膜缺血再灌注损伤Fas/FasL的表达与细胞凋亡的关系

目的:探讨Fas/FasL在大鼠视网膜缺血再灌注损伤(retinal ischemia reperfusion injury,RIRI)中的表达与细胞凋亡的关系及α-硫辛酸(α-LA)的治疗作用。方法:采用升高眼内压的方法建立RIRI模型,将54只SD大鼠随机分为正常组、缺血再灌注(IR)组和α-LA干预组,IR组及α-LA干预组在12,24,48,72h光镜观察视网膜内层厚度及神经节细胞丢失,TUNEL法检测视网膜神经细胞凋亡,免疫组织化学法检测视网膜组织Fas/FasL蛋白的表达。结果:IR组视网膜内层变薄,神经节细胞数目减少。细胞凋亡出现于再灌注后12h,并逐渐递增,24h达到高峰,48h开始下降。Fas/FasL表达与凋亡的神经细胞改变基本一致。α-LA干预组视网膜内层厚度和神经节细胞数目有一定恢复。凋亡细胞在12,24,48h组明显低于IR组(P<0.05);Fas表达在12,24h组较IR组显著下降(P<0.05);FasL表达在12,24,48h组较IR组明显下降(P<0.05)。结论:Fas/FasL系统参与RIRI,导致神经节细胞凋亡;α-LA可下调Fas/FasL表达,减少神经节细胞凋亡。     

内毒素诱导的大鼠葡萄膜炎细胞凋亡的研究

目的探讨大鼠内毒素诱导的葡萄膜炎（EIU）中葡萄膜和视网膜组织浸润细胞的凋亡,及Fas-FasL的表达与细胞凋亡的相关性。方法注射霍乱弧菌内毒素诱发大鼠EIU模型,内毒素注射后6、12、18、24、48h进行临床观察评分并行组织病理学检查。应用TUNEL法检测葡萄膜和视网膜中的凋亡细胞,免疫组织化学法检测Fas和FasL在葡萄膜和视网膜中的表达和变化。结果实验组大鼠均发生了EIU;葡萄膜和视网膜组织浸润细胞中可见TUNEL染色阳性凋亡细胞,18h达高峰,以虹膜和睫状体最为明显,凋亡阳性细胞数与正常对照组相比,差异有统计学意义（P〈0.01）;实验各组中均可见Fas表达,实验各组中FasL表达显著增强,与正常对照组相比差异有统计学意义（P〈0.01）。Fas和FasL的表达与凋亡之间呈正相关（r=0.923,0.807,P〈0.01）。结论EIU中葡萄膜和视网膜的浸润细胞存在凋亡现象,凋亡参与了炎症的迅速消退,Fas和FasL的高表达可能与内毒素诱导的葡萄膜炎眼组织的细胞凋亡有关。     

Apoptosis in proliferative vitreoretinopathy

PURPOSE: To study the involvement of apoptosis using different apoptosis markers in PVR pathogenesis. METHODS: The presence of mRNA coding for Fas, Fas ligand (FasL), and TNF-related apoptosis inducing ligand (TRAIL) was investigated in vitreous samples from 46 consecutive patients-25 with PVR, 11 with retinal detachment (RD) not complicated by PVR, and 10 with macular hole (MH)-using RT-PCR. From previously examined vitreous samples, 21 PVR, 9 RD, and 10 MH were examined for their levels of TGF-beta2 protein with sandwich ELISA kits. Five epiretinal membranes excised from five patients with PVR were also examined for apoptotic cell death using the terminal deoxytransferase (TdT) mediated dUTP-biotin nick end labeling (TUNEL) technique. RESULTS: FAS mRNA was detected in 72% of patients with PVR, 55% of patients with RD and 20% of patients with MH. TRAIL mRNA was detected in 67% of patients with PVR, 89% of patients with RD, and 20% of patients with MH. FasL mRNA was detected in 20% of patients with PVR, 9% of patients with RD, and 10% of patients with MH. The median levels of Fas and TRAIL mRNA were significantly higher (P < 0.05) in patients with PVR than in those with MH hole but between patients with PVR and those with RD the difference was not significant (P > 0.05). A significant difference was detected between RD and MH for TRAIL mRNA levels (P = 0.008). For FasL, no significant difference between groups was found. TGF-beta2 was detected in all investigated vitreous samples. A significant difference was found between the PVR and MH groups (P = 0.001) and between the RD and MH groups (P = 0.004), but not between the PVR and RD groups (P < 0.05). The level of TGF-beta2 was significantly correlated to the level of TRAIL mRNA (r = 0.86), but no correlation was found between TGF-beta2 and Fas mRNA levels (r = 0.21). Four of five examined PVR epiretinal membranes showed positive staining for apoptotic cells using the TUNEL technique. CONCLUSIONS: Apoptosis is one of the mechanisms that is involved in PVR pathogenesis. Different apoptosis markers suggest different pathways occur in PVR, including Fas/FasL, TRAIL, and TGF-beta2 mediated processes.     

Fas/FasL抗原在Behcet病患者外周血T淋巴细胞的表达

目的研究Fas/FasL抗原在Behcet病患者外周血T淋巴细胞的表达及其可能的意义。方法采用流式细胞仪(flowcytometry)及三色荧光双标记细胞免疫荧光技术对26例Behcet病患者和43例正常人外周血T淋巴细胞表面Fas和FasL抗原的表达进行检测。结果Behcet病CD4+T细胞Fas表达阳性率为25.70%±7.32%,对照组为14.02%±6.30%,二者有显著性差异(P＜0.01);但CD4+T细胞的FasL表达在2组间无显著性差异。CD8+T细胞Fas表达阳性率为9.47%±6.97%,对照组为3.47%±2.75%,二者有显著性差异(P＜0.01),但FasL表达阳性率二者无显著性差异。结论Behcet病患者外周血CD4+和CD8+T细胞Fas的表达阳性率高于正常对照组,FasL的表达阳性率相对不足,提示这些活化的Fas+T淋巴细胞不能有效地通过Fas/FasL系统介导的凋亡途径被清除,此可能是Behcet病患者体内大量活化的淋巴细胞长期存在和炎症持续存在及复发的重要原因之一。     

Fas/FasL系统在大鼠视网膜缺血再灌注凋亡损伤中的作用及bFGF的影响

目的：探讨Fas／FasL在大鼠视网膜缺血再灌注损伤中的表达与细胞凋亡的关系及碱性成纤维细胞生长因子(basic fibroblast growth Factor,bFGF)的影响。方法：前房加压法制作大鼠视网膜缺血再灌注损伤模型，28只大鼠随机分为正常组和手术组，其中手术组大鼠左眼为缺血再灌注组，右眼为治疗组，手术组又按照再灌注后不同时间段分为1，6，12，24，48，72h组。应用末端脱氧核酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法检测视网膜神经细胞凋亡指数(apoptosis index，AI)，免疫组织化学法检测视网膜组织中Fas，FasL的表达。结果：视网膜神经细胞的凋亡出现于再灌注后6h，并逐渐递增，24h达到高峰，48h开始下降，72h组不明显。Fas，FasL表达改变与凋亡的神经细胞改变基本一致。bFGF治疗组各观察指标表达变化规律与缺血组基本一致，但AI值在12，24，48h组明显低于缺血组(P＜0．05)；Fas表达在6，12，24h组较缺血组显下降(P＜0．05)；FasL表达在12，24，48h组较缺血组明显下降(P＜0．05)。结论：Fas／FasL死亡诱导系统参与视网膜缺血再灌注损伤，导致神经节细胞的凋亡；bFGF可抑制Fas、FasL的表达，减少神经节细胞凋亡，对视网膜缺血再灌注损伤有治疗作用。     

Fas mediates apoptosis and oxidant-induced cell death in cultured hRPE cells

PURPOSE: To investigate whether Fas ligand (FasL) and the Fas receptor system mediates apoptosis in cultured human retinal pigment epithelial (hRPE) cells and contributes to oxidant-induced death of hRPE cells. METHODS: Expression of FasL and Fas in cultured hRPE cells was examined by Western blot analysis and flow cytometry. The susceptibility of hRPE cells to Fas-mediated apoptosis was determined by incubating cells with recombinant soluble Fas ligand (sFasL). Characteristics of apoptosis assessed included chromatin condensation, DNA cleavage, and phosphatidylserine exposure. To investigate the possible involvement of Fas-mediated apoptosis in oxidative killing of hRPE cells, the effects of the oxidant tert-butylhydroperoxide (tBH) on the expression of FasL and Fas were studied. The specificity of effects of oxidant was examined using the antioxidants glutathione and N-acetyl-L-cysteine (NAC). The requirement for the Fas pathway in tBH-induced apoptosis was investigated using an antagonistic anti-Fas antibody ZB4 that blocks the interaction between FasL and Fas. RESULTS: Cultured hRPE cells constitutively expressed FasL and Fas. Ligation of Fas receptor with recombinant sFasL triggered apoptosis in hRPE cells. tBH treatment of hRPE cells resulted in increased expression of FasL and Fas. Glutathione and NAC completely abrogated tBH-induced increase in FasL and Fas expression and apoptosis. Blocking FasL and Fas interaction by ZB4 inhibited tBH-induced apoptosis, but only partially. CONCLUSIONS: A functional Fas-mediated apoptotic pathway is present in cultured hRPE cells and can be activated with sFasL or by upregulation of FasL and Fas expression with an oxidant. The incomplete inhibition by blocking antibody indicates that the Fas pathway is involved in oxidant-induced apoptosis, but other triggering mechanisms are also important.     

Kinetics of apoptotic cells in experimental autoimmune uveoretinitis

PURPOSE: To investigate the role of apoptosis in immunopathogenic mechanisms of experimental autoimmune uveoretinitis (EAU), the kinetics of apoptotic cells and expression of Fas and Fas ligand (FasL) in the eye with EAU were studied. METHODS: Male inbred Lewis rats were immunized with S-antigen (40 microg/rat), and eyes were examined to detect apoptotic cells on days 1, 4, 8, and 10 post-immunization and days 0, 2, 4, 6, and 8 after the onset of EAU. The clinical and pathologic scores were used for estimating EAU. Apoptotic cells were analyzed by TdT-mediated dUTP nick-end labeling, electron microscopic and immunohistologic examinations, and agarose gel electrophoresis. The anti-rat Fas and anti-rat FasL antibodies were used to examine the expression of Fas and FasL. RESULTS: Apoptotic cells were detected in the infiltrating cells in the aqueous humor, the vitreous body, the iris-ciliary body, and the retina. Apoptotic cells were observed as early as the day of EAU onset and reached a peak on day 2 after the disease onset. Fas and FasL were expressed on the infiltrating cells in the aqueous humor and the vitreous. FasL was expressed on resident cells of the ciliary body. The kinetics of the expression of FasL corresponded with the kinetics of apoptotic cells. CONCLUSIONS: Fas-FasL-mediated apoptosis is considered to occur in the eye with EAU and plays a role in the immunopathogenic mechanisms to eliminate ocular infiltrating cells, thereby down-regulating the inflammatory processes.     

Influence of IL-1β and TNF-α on Fas Expression of Human Retinal Pigment Epithelial Cells in Vitro

Purpose: To observe Fas expression change of cultured human retinal pigment epithelial (RPE) cells by IL-lβ and TNF-α. Methods: With flow cytometry, immunohischemistry, and color imaging system, Fas expressions by exposure to IL-1β and/or TNF-α were measured. Results:The gray degree values of Fas expression were 67.5 ±6.1 in IL-1β+TNF-α-treated group, 80.1 ±9.2 in IL-1 β-treated group, and 70.4±6.4 in TNF-α-treated group, respectively. There were significant differences (P ＜ 0.005) compared with control group (107.0±10.2). Flow cytometry showed that 15.0% cultured human RPE cells expressed Fas. Fas-positive in IL-lβ, TNF-α, and IL-lβ+TNF-α-treated groups expressed was 28.1%, 34.5%, and 65.2%, respectively. Conclusion: IL-1 β, TNF-α, and combining both of them can up-regulate Fas protein expression, which may contribute to more Fas (+) cells in proliferative vitreoretinopathy PVR). Minimizing this process by means of inducing apoptosis of Fas (+) proliferative cells of Fas/FasL pathway is a future preventive and therapeutic possibility for PVR. Eye Science 2004;20:39-41.     

Influence of IL-1 beta and TNF-alpha on Fas expression of human retinal pigment epithelial cells in vitro.

PURPOSE: To observe Fas expression change of cultured human retinal pigment epithelial (RPE) cells by IL-1 beta and TNF-alpha. METHODS: With flow cytometry, immunohischemistry, and color imaging system, Fas expressions by exposure to IL-1 beta and/or TNF-alpha were measured. RESULTS: The gray degree values of Fas expression were 67.5 +/- 6.1 in IL-1 beta + TNF-alpha-treated group, 80.1 +/- 9.2 in IL-1 beta-treated group, and 70.4 +/- 6.4 in TNF-alpha-treated group, respectively. There were significant differences (P < 0.005) compared with control group (107.0 +/- 10.2). Flow cytometry showed that 15.0% cultured human RPE cells expressed Fas. Fas-positive in IL-1 beta, TNF-alpha, and IL-1 beta + TNF-alpha-treated groups expressed was 28.1%, 34.5%, and 65.2%, respectively. CONCLUSION: IL-1 beta, TNF-alpha, and combining both of them can up-regulate Fas protein expression, which may contribute to more Fas (+) cells in proliferative vitreoretinopathy (PVR). Minimizing this process by means of inducing apoptosis of Fas (+) proliferative cells of Fas/FasL pathway is a future preventive and therapeutic possibility for PVR.