SLE患者外周血白细胞凋亡相关基因Fas,sFas mRNA表达研究

利用荧光标记ＲＴ－ＰＣＲ半定量方法，对ＳＬＥ患者，ＲＡ患者和正常人外周血白细胞凋亡相关基因Ｆａｓ、ｓＦａｓ ｍＲＮＡ表达水平进行了检测。结果发现：ＳＬＥ患者Ｆａａｓ、ｓＦＡ表达高于正常人（Ｐ〈０．０００１和Ｐ〈０．００１），并且表达量与病情活动度相关，而ＲＡ患者仅ｓＦａｓ表达增高（Ｐ〈０．０１），这提示凋亡基因表达异常在ＳＬＥ发病中可能具有一定作用和意义。     

Fas系统在视网膜母细胞瘤的表达

目的 观察视网膜母细胞瘤（ＲＢ）中Ｆａｓ和Ｆａｓ配体（ＦａｓＬ）的表达特征。方法 应用免疫组化法检测３２例ＲＢ标本中Ｆａｓ和ＦａｓＬ的表达。结果 Ｆａｓ和ＦａｓＬ在ＲＢ中的阳性表达率分别为７０．６％和６５．８％,二者呈正相关（Ｐ〈０．００１）。分化程度低的和经过放疗的表达率明显增高（Ｐ〈０．０５或０．０１）。其表达率与视神经受浸润与否、眼内期或青光眼期无关（Ｐ〉０．０５）。结论 ＲＢ中Ｆａｓ及ＦａｓＬ高表达,分化程度越低表达越强。Ｆａｓ和ＦａｓＬ的表达可作为临床判断ＲＢ恶性程度的指标之一。     

兔眼小梁切除术后滤过泡组织Fas mRNA的表达

目的　对小梁切除术后滤过泡组织ＦａｓｍＲＮＡ的表达进行初步研究。方法　新西兰白兔４８只单独笼养,随机选取４０只兔右眼行小梁切除术,使上方结膜形成滤过泡,按术后时间又分为１ｄ、７ｄ、１４ｄ、２０ｄ、２８ｄ共５个亚组,每组８只兔。另外８只兔的右眼未进行手术为正常对照组。在行小梁切除术后１ｄ、７ｄ、１４ｄ、２０ｄ、２８ｄ,切下滤过手术区组织,同时于２８ｄ时切除正常对照组相应部位的结膜组织。实时荧光定量ＰＣＲ检测各组标本的ＦａｓｍＲＮＡ表达情况。结果　兔眼小梁切除术后１ｄ、７ｄ、１４ｄ三个亚组ＦａｓｍＲＮＡ的相对表达量分别为１．３３±０．３５、１．４１±０．２８、１．３８±０．２５,与正常对照组的表达量１．４２±０．３４相比差异无统计学意义（Ｐ＞０．０５）,术后２０ｄＦａｓｍＲＮＡ相对表达量为０．９０±０．１８,较对照组明显下降,差异有统计学意义（Ｐ＜０．０５）,２８ｄ时进一步下降至０．７８±０．０９（Ｐ＜０．０５）。结论　在兔眼结膜滤过泡组织愈合过程中,术后２０ｄ及２８ｄ凋亡主要基因ＦａｓｍＲＮＡ表达量明显减少,可能为减少青光眼小梁切除术后瘢痕化寻找到一个新的突破口。     

兔准分子激光角膜切削术后角膜血小板源性生长因子mRNA表达的变化

研究角膜上皮下混浊形成的发生机制,检测准分子激光屈光性角膜切削术后角膜上皮和基质血小板源性生长因子表达的变化。方法新西兰白兔施行ＰＲＫ后１,２,３月用裂隙灯显微镜观察ｈａｘｅ形成情况,并用原位酸分子杂交方法,检测角膜上皮和基质ＰＤＧＦ ｍＲＮＡ的表达。结果正常角膜上皮细胞有ＰＤＧＦ ｍＲＮＡ表达,基质层无表达;ＰＲＫ后角膜上皮细胞ＰＤＧＦｍＲＮＡ表达增加,术后２月表达最强,且基质中亦有轻微表达。上     

角膜移植中Fas／FasL系统的研究进展

角膜移植排斥反应是导致植片失败的主要原因。本文主要介绍了Ｆａｓ／Ｆａｓ ｌｉｇａｎｄ（ＦａｓＬ）系统相互作用诱导细胞凋亡后产生的对异体植片的保护作用及其机制探讨。     

Fas和FasL及其在眼科的研究进展

自１９８９年和１９９３年分别获得Ｆａｓ和Ｆａｓ配体（ＦａｓＬ）以来，其在生物医学各个领域得到广泛而深入的研究。Ｆａｓ分子为典型的Ｉ型膜蛋白，胞内段存在传递死亡信号的死亡域。ＦａｓＬ属于ＴＮＦ家庭成员，其结合到Ｆａｓ分子以后，通过死亡域传递细胞凋亡信号，引起Ｆａｓ表达的细胞死亡。本文就Ｆａｓ／ＦａｓＬ基因、蛋白结构和功能以及在产方面的研究作一简要综述。     

Fas和FasL介导的调亡在角膜移植中的作用

Ｆａｓ与ＦａｓＬ介导的凋亡可能在以下三个方面对角膜移植术的成功产生影响：１．抑制新生血管的形成，降低角膜移植的高危因素。２．影响前房相关免疫偏离，维持眼部的免疫赦免地位。３．阻止炎性细胞渗透，减少免疫排斥反应对局部组织的损害。深入了解这一系统的作用机制并利用其提高角膜移植术的成功率，具有很大的临床用价值。     

羊膜对激光角膜切除术后TGF—β1表达调控的初步研究

目的 探讨羊膜对激光角膜光学切除术（Ｐｈｏｔｏｒｅｆｒａｃｔｉｖｅ ｋｅｒａｔｅｃｔｏｍｙ ＰＲＫ）后角膜组织ＴＧＦ－β１表达的影响。方法 对６只新西兰白兔双眼行ＰＲＫ,术后１眼立即行角膜表面羊膜移植术,只眼作为对照。术后４周用原位杂交方法检测角膜上皮和基质ＴＧＦ－β１ｍＲＮＡ的表达。结果 ＰＲＫ后４周对照组角膜上皮和基质有较浓度的ＴＧＦ－β１ｍＲＮＡ表达,羊膜移植组角膜上皮和基质亦有ＴＧＦ－β１     

纤维介素蛋白2（FGL-2）在大鼠角膜移植排斥反应中的作用

目的　研究纤维介素蛋白２（ｆｉｂｒｉｎｏｇｅｎ-ｌｉｋｅｐｒｏｔｅｉｎ２,ＦＧＬ-２）在大鼠角膜移植排斥反应发生发展过程中的作用及糖皮质激素对其表达的影响。方法　６０只成年雌性Ｗｉｓｔａｒ大鼠随机分为正常对照组、自体角膜移植组、同种异体角膜移植组和糖皮质激素组。正常对照组大鼠未行任何手术,自体角膜移植组、同种异体角膜移植组及糖皮质激素组均行穿透性角膜移植术。术后裂隙灯显微镜下观察术眼角膜炎性反应情况,按照Ｌａｒｋｉｎ的标准进行排斥反应评分,计算排斥反应指数,并在术后９ｄ和１４ｄ分别取眼球制备角膜组织切片,行组织病理学检查和免疫组织化学检测,观察角膜组织的炎性反应和ＦＧＬ-２在角膜组织中的表达;采用实时荧光定量ＰＣＲ法检测ＦＧＬ-２ｍＲＮＡ在大鼠角膜植片中的相对表达量。结果　术后９ｄ内各组角膜植片均出现不同程度的水肿、混浊及新生血管生长。同种异体角膜移植组大鼠平均在角膜移植术后９．８ｄ发生排斥反应并获病理学支持;自体角膜移植组和糖皮质激素组大鼠术后排斥反应计分均未达到诊断标准。免疫组织化学结果显示,不同时间点同种异体角膜移植组炎性细胞明显多于自体角膜移植组和糖皮质激素组。术后３组手术干预组角膜ＦＧＬ-２ｍＲＮＡ的表达均增加,在术后９ｄ和１４ｄ时同种异体角膜移植组均较自体角膜移植组和糖皮质激素组高,差异均有统计学意义（均为Ｐ＜０．０５）。结论　ＦＧＬ-２可能参与角膜移植免疫排斥反应,糖皮质激素可能通过降低其表达发挥抗排斥作用。     

FasL,pRb在视网膜母细胞瘤中表达的研究

目的 探讨ＦａｓＬ及ｐＲｂ在视网膜母细胞中的表达及意义。方法 用免疫组织ＳＰ方法检测４５例视网膜母细胞瘤及１２例正常视网膜组织中ＦａｓＬ及ｐＲｂ蛋白的表达。结果 网膜母细胞瘤细胞表达ｐＲｂ、比正常组织显著降低。视网膜母细胞瘤和正常组织均高表达ＦａｓＬ。结论ＦａｓＬ的表达是贯穿视网膜细胞铰琢发展过程中的一个重要事件,ｐＲｂ低表达与肿瘤的发生有密切关系。     

Immunohistochemical Expression of Fas Ligand (FasL) and Neprilysin (Neutral endopeptidase/CD10) in Keratoconus

Recent evidence demonstrated a correlation between apoptosis and neprilysin expression. The aim of this study was to investigate the immunohistochemical expression of Fas ligand (FasL) and neprilysin in keratoconic corneas in comparison to normal cadaver corneas to evaluate if such molecules play a role in the pathogenesis of keratoconus. We studied the expression of FasL and neprilysin in corneal specimens removed during penetrating keratoplasty in 15 cases with keratoconus and compared them with 5 normal cadaver corneas. In keratoconus, FasL was expressed in epithelium, endothelium and sub-Bowman’s stroma only, while neprilysin was expressed in epithelium, endothelium and all stromal layers. All normal corneas showed weak expression of both markers in basal epithelial layer only. In keratoconus, corneal epithelium with higher expression of FasL may evoke apoptosis in keratocytes, while neprilysin could prevent possible rescue of keratocytes from apoptosis.     

Non-cleavable mutant Fas ligand transfection of donor cornea abrogates ocular immune privilege

The expression of Fas ligand (FasL) in donor corneal tissue has been thought to contribute to the prolonged survival of orthotopic corneal allografts. However, in solid organ transplantation, FasL gene-transfected tissues have been reported to lead to graft destruction through neutrophil recruitment. We wished to examine whether transfection of mutant FasL cDNA, which produces only membrane-binding forms of FasL because of its cleavage site, prolonged corneal allograft survival in mice. Donor corneal tissues were transduced with two adenovirus vectors containing human mutant FasL cDNA (AxCALNmFasL) and Cre recombinase (AxCANCre). Donor corneal tissues were then transplanted orthotopically onto recipient mice, and graft clarity was examined by slit lamp microscopy. We found that donor corneal grafts transfected with mutant FasL were intensely opaque for 1-2 weeks after grafting, when grafts without transfection remained clear. Histological examination revealed polymorphonuclear cell infiltration in the transfected grafted tissue. Moreover, mutant FasL transduced to donor tissues was found to exert its effects by binding to host, rather than donor Fas molecules, since corneal grafts with mutant FasL survived as long as those without transfection only when host animals, but not donor animals, lacked Fas molecules. Our results indicate that membrane-binding FasL over expression in donor cornea does not prolong corneal allograft survival; indeed, it causes rapid graft destruction.     

The expression of Fas ligand protein in ultraviolet-exposed rabbit corneas.

PURPOSE: Keratocytes undergo apoptosis during various pathologic conditions and after exposure to ultraviolet radiation (UVR). It was reported that the Fas/Fas ligand system is involved in modulating keratocyte apoptosis. The expression of Fas ligand (FasL) protein was studied in rabbit corneas after photokeratitis induced by different UV wavelengths. METHODS: Six New Zealand albino rabbit corneas were exposed to 280- and 310-nm UVR in 10-nm full wavebands at doses producing biomicroscopically significant keratitis (0.12 J/cm2 for 280 nm and 0.47 J/cm2 for 310 nm). Animals were killed 24 hours after exposure. Immunohistochemistry was performed to localize FasL protein in paraffin sections of rabbit corneas. Primary antibody was polyclonal goat anti-FasL IgG. RESULTS: FasL protein was uniformly detected in epithelial and endothelial layers of all UVR-exposed and control, nonexposed corneas. The positive staining of keratocytes was confined to the anterior stroma of corneas that were exposed to 280-nm UVR. Corneas exposed to 310-nm UVR showed positively stained keratocytes throughout the entire thickness of the stroma. CONCLUSIONS: These data strongly suggest that the Fas/FasL system may play an important role in apoptosis of corneal cells after UVR. The FasL expression in the corneal stroma was more extensive after exposures at 310-nm UVR than at 280-nm UVR.     

Effect of metalloprotease inhibitors on corneal allograft survival

PURPOSE: The expression of Fas ligand (FasL) in the cornea is essential for corneal allograft acceptance in mice. Because the expression of FasL on the surface of cells is sensitive to cleavage with matrix metalloproteases (MMPs), this study examined whether inhibitors of MMPs would lead to increased FasL expression and improved corneal allograft survival. METHODS: Corneal endothelia derived from mice and humans were treated with MMP inhibitors, and FasL expression was examined. BALB/c mice were engrafted with C57BL/6 or C57BL/6-gld corneas and treated with an ointment containing the MMP inhibitor, doxycycline. Corneal allograft survival was monitored for 50 days. RESULTS: Corneal endothelial cells from both mice and humans displayed increased surface expression of FasL after treatment with MMP inhibitors. The increase in surface expression was further evidenced by the ability of these cells to kill Fas-expressing target cells. Mice treated with doxycycline after corneal allograft transplantation showed significantly prolonged allograft survival and an increase in the overall acceptance rate. CONCLUSIONS: MMP inhibitor treatment of cornea-derived endothelial cells results in increased FasL expression and function. MMP inhibitor treatment prolongs corneal allograft survival and results in a modest increase in corneal allograft acceptance.     

胎儿角膜深低温保存对FAS/FASL蛋白表达的影响

目的:研究细胞凋亡信号分子Fas和细胞凋亡因子FasL在新鲜及深低温保存后胎儿角膜的表达情况。方法:应用免疫组织化学SABC法对33例不同胎龄(5mo至足月)胎儿角膜石蜡标本切片后,分别进行了Fas和FasL蛋白染色。结果:胎儿角膜细胞Fas蛋白及FasL蛋白全为胞质和/或胞膜着色。33/33例角膜上皮细胞、基质细胞及内皮细胞Fas蛋白阳性表达,经统计学分析,未发现不同月龄间有差异。33/33角膜上皮细胞及内皮细胞FasL蛋白阳性表达,经统计学分析,未发现不同月龄间有差异,角膜基质细胞FasL蛋白的表达为0/33。新鲜角膜和冷冻保存后角膜的Fas,FasL蛋白阳性表达量经统计学分析无差别。结论:新鲜和深低温保存后的5mo至足月龄胎儿角膜均可见Fas,FasL蛋白的表达。其中Fas在角膜上皮、基质和内皮细胞均表达,FasL在角膜上皮、内皮细胞表达,而基质细胞不表达。深低温保存对胎儿角膜Fas/FasL蛋白表达无影响。     

接合黏附分子-1在大鼠角膜中的表达研究

背景 接合黏附分子-1(JAM-1)是新发现的跨膜蛋白,参与细胞紧密连接的结构组成和功能发挥.在眼组织方面,紧密连接对维持角膜的透明性十分重要,但是目前就JAM-1在角膜紧密连接结构和功能方面的研究较少. 目的 确定JAM-1在大鼠角膜上皮层、基质层和内皮层的构成.方法 选取4只SPF级Wistar大鼠,2只用于JAM-1基因在角膜组织中表达的逆转录聚合酶链反应(RT-PCR)检测,另2只用于免疫组织化学检测.动物过量麻醉处死后获得角膜组织并制备角膜上皮、基质和内皮标本,RT-PCR法检测角膜标本中JAM-1、occludin和claudin-1 mRNA的表达.反应产物行质量分数1.5％琼脂糖凝胶电泳并用凝胶成像系统进行分析.用兔抗鼠JAM-1单克隆抗体对角膜石蜡切片、上皮及内皮铺片行免疫组织化学检测,评估JAM-1蛋白在大鼠角膜组织各层的表达部位和表达强度. 结果 在大鼠角膜各层均可检测到JAM-1、occludin和claudin-1 mRNA的表达,PCR熔解曲线为清晰的单峰.角膜组织各层中JAM-1 mRNA表达水平与occludin mRNA相似,均高于claudin-1 mRNA.3种黏附分子均在上皮层表达最强,角膜基质层表达较弱.免疫组织化学检测显示,JAM-1蛋白在角膜各层均有明确的阳性染色,角膜上皮基底层的表达强于基质层和内皮层.角膜上皮、内皮铺片检测显示,JAM-1蛋白主要表达于上皮细胞和内皮细胞的连接部位,而角膜内皮中JAM-1蛋白的阳性染色广泛而弥散.结论 JAM-1作为细胞连接的构成成分,在角膜上皮层、内皮层和基质层均有表达,但其表达的形态和水平因组织层次的不同而不同.     

HLA antigenicity of normal and pathological corneas

Fresh human corneas and corneal buttons were studied for expression of HLA antigens. Using monoclonal antibodies in an indirect immunofluorescence assay, corneal layers were examined for class I (HLA-A, B, C) and class II (HLA-DR) histocompatibility antigens. Twenty-one human corneas were studied, 6 normal and 15 pathological: 4 buttons of allograft rejection, 9 buttons of pseudophakic bullous keratopathy. In fresh control corneas, HLA-A, B, C antigens were localized on corneal epithelium and on stromal keratocytes but were never found on endothelial cells. HLA-DR antigens were not detected on corneal epithelium, stroma or endothelium but were detected on Langerhans cells within epithelium and anterior stroma. At the corneal limbus, HLA class I-II antigens were expressed on vascular endothelium. HLA antigen distribution was modified in pathological corneas. Antigens HLA-A, B, C were induced on endothelial cells of rejected corneal allografts. Antigens HLA-DR were detected on epithelial cells, cells in the stroma of pseudophakic bullous keratopathy and also on endothelial cells of rejected corneal allografts. These results suggest that induction of class I and II antigen expression by inflammatory factors may occur in vivo. In rejected corneal allografts induction of HLA-DR antigen on corneal layers would intensify the process of rejection. This study and others have demonstrated the ability of modulation of HLA antigen expression on human corneal cells in vivo.     

大鼠高危角膜移植免疫排斥反应中Fas/FasL表达

目的：研究大鼠高危角膜移植免疫排斥反应的病理变化和Fas／FasL的表达及IL-lra对Fas／FasL表达的影响。方法：用40只Wistar大鼠作为受体，20只SD大鼠作为供体，用缝线法诱导受体大鼠角膜新生血管，建立高危角膜移植动物模型。将实验大鼠随机分为实验组和对照组，每组10只。实验组从术后第一天起用IL-lra 5mg／ml点眼治疗，4次／天，共30天。对照组用相同的方法生理盐水点眼治疗。应用免疫组织化学染色方法检测IL—1ra治疗组和生理盐水对照组术后不同时期角膜植片中Fas和FasL的表达。结果：角膜移植术后Fas／FasL的表达明显升高，IL-1ra可对此产生明显的抑制作用。结论：Fas／FasL与角膜移植免疫排斥反应密切相关，IL—1ra可以抑制角膜移植免疫排斥反应中Fas／FasL的表达。     

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。     

Behcet病周围血淋巴细胞Fas和FasL的表达

目的 观察Behcet病周围血淋巴细胞Fas和FasL的表达及其意义。方法 应用Oligod(T)18和M-MuLv逆反录酶方法，观察19例Behect病患者和19个正常人周围血淋巴细胞的Fas和FasL mRNA表达，并用流动细胞计和免疫荧光法进一步观察T淋巴细胞的Fas和FasL的表达。结果 Behcet病组Fas和FasL的表达明显高于对照组。Fas抗原在CD4^ 和CD86 的表达明显高于对照组，但FasL的表达与对照组无明显表达明显增设，但其分子表达不平衡，此可能是Behcet病慢性和复发性炎症的一个重要机制。