Toll样受体-4在葡萄膜炎中的表达和作用

Toll样受体-4(TLR4)作为内毒素的识别受体在固有免疫与适应性免疫之间的桥梁作用越来越受到重视.葡萄膜炎发病中TLR4的作用不断被证实:TLR4基因突变的C3H/HeJ小鼠不能诱导出葡萄膜炎;正常人眼葡萄膜中有TLR4表达而且是有功能的;葡萄膜炎患者外周血中单核细胞TLR4功能下降.本文对TLR4在葡萄膜炎发病中的信号传导通路的进展及其在抗感染中的作用进行了综述.     

大鼠视神经损伤后Toll样受体4在视网膜中的表达

背景Toll样受体4（TLR4）是一种重要的免疫相关受体,在多种疾病的发生中起致炎作用。研究发现,视神经损伤后继发的炎症反应可进一步引起视网膜损伤,因此视神经损伤后TLR4的表达及其效应值得研究。目的研究大鼠视神经损伤后视网膜TLR4的表达情况。方法选取成年健康SPF级SD大鼠24只,按随机数字表法随机分为视神经损伤3d组和视神经损伤7d组。取大鼠右眼用视神经钳夹法制备视神经损伤模型,左眼不予处理为对照组。分别于视神经损伤后3d和7d用过量麻醉法处死大鼠并分离视网膜,采用免疫荧光法检测各组大鼠视网膜中TLR4的表达;分别采用逆转录PCR法（RT—PCR）和Westernblot法检测大鼠视网膜中TLR4mRNA及其蛋白的表达;采用TUNEL染色法观察各组大鼠视网膜神经节细胞（RGCs）的凋亡情况。结果视网膜免疫荧光法检测结果显示,TLR4在大鼠视网膜中呈绿色荧光,视神经损伤3d组和视神经损伤7d组造模眼视网膜中的荧光强度较对照组左眼均明显增强,绿色荧光主要分布在视网膜内层。RT—PCR法检测表明,模型眼视网膜损伤后3d和7d视网膜中TLR4mRNA相对表达量分别为2．92±0．06和3．92±0．12,对照眼TLR4mRNA的相对表达量分别为2．87±0．12和3．44±0．17,大鼠模型眼TLR4mRNA表达的灰度值较对照眼明显增加,差异均有统计学意义（t3d=-12．888,P〈0．001;t7d=-4．669,P=0．010）。Westernblot法检测显示,大鼠模型眼视网膜损伤3d和7d视网膜中TLR4蛋白的相对表达量分别为1．14±0．05和1．49±0．03,对照眼TLR4蛋白的相对表达量分别为0．99±0．09和1．38±0．07,模型眼视网膜中TLR4蛋白表达量明显高于对照眼,差异均有统计学意义（t3d=-11．324,P〈0．001;t7d=-5．638,P=0．005）。TUNEL染色显示,模型眼RGCs凋亡数较对照眼增多。结论TLR4在视神经损伤大鼠视网膜内层的表达明显上调,提示TLR4通路可能参与RGCs的损伤。     

Toll样受体在眼部各组织中的表达及其作用

Toll样受体是新近发现的一类广泛存在于植物、昆虫和哺乳动物体内的与跨膜信号转导有关的受体，在进化上具有高度保守性。研究揭示了其在机体免疫特别是抗感染免疫中有重要作用。越来越多的研究已经发现人眼角膜细胞、巩膜、结膜、葡萄膜、视网膜、虹膜内皮细胞、睫状体等都能表达Toll样受体，而且与眼部的免疫和炎症反应密切相关。Toll样受体的发现为深入研究眼部感染性疾病的发病机制及临床治疗开辟了更为广阔前景。     

Toll样受体在干眼发病机制中的研究进展

最近研究显示眼表引发免疫炎症反应的部分原因是眼表存在一类称为Toll样受体（TLRs）的蛋白家族。TLRs在眼部的表达受到感染和各种炎症环境的影响,例如单疱病毒、细菌、真菌性角膜炎,变态反应性结膜炎和干眼综合征。TLRs信号通路对抗原递呈细胞和T细胞介导的免疫炎症反应的激活起重要作用。近些年随着眼科学的发展,对TLRs在干眼发病机制方面的研究逐渐增多,现就TLRs的结构与配体,TLRs的信号转导路径,TLRs在眼表中的表达位置及表达模式,以及与干眼的相关性作一综述。     

Toll样受体在眼科的研究现状

Toll样受体是自身免疫的关键受体，它参与宿主对致病微生物的识别和防御等感染免疫过程，也与人体的获得性免疫密切相关，是目前免疫学研究的重点和热点。眼球作为感染性和免疫性疾病的好发部位，在感染免疫和获得性免疫方面与全身摹他器官有相似之处，但是其独特的解剖特点，例如血房水屏障、血视网膜屏障、角膜的无血管化等，使其免疫学特点的特异性也非常明显。就Toll样受体的特点以及在眼科的研究现状进行综述。     

Toll样受体及VEGF因子在不同类型翼状胬肉中的表达和意义

目的 比较Toll样受体(TLR2、TLR3、TLR4)及血管内皮生长因子(VEGF)在初发性和复发性翼状胬肉中的表达差异:设计 实验研究。研究对象 初发翼状胬肉组织36例,复发翼状胬肉组织24例,正常结膜组织20例。方法 免疫组化法检测TLR2、TLR3、TLR4、VEGF及肿瘤坏死因子(TNF)-α在正常结膜、初发及复发性翼状胬肉组织中的表达情况。分析VEGF与TLR2、TLR3、TLR4表达的相关性。主要指标 TLR2、TLR3、TLR4、VEGF及TNF-α的表达量、阳性表达率及相关系数。结果 TLR2、TLR3、TLR4在正常结膜组、初发性翼状胬肉和复发性翼状胬肉的表达量依次上调。复发性翼状胬肉的间质层中存在大量VEGF棕黄色颗粒,上皮层中也有表达。VEGF与TLR2、TLR3和TLR4在复发性翼状胬肉中表达呈正相关(r=0.512,P=0.001;r=0.671,P=0.001;r=0.482,P=0.001)。结论 复发性翼状胬肉中TLR2、TLR3、TLR4、VEGF因子的表达高于初发性翼状胬肉,提示免疫炎症和VEGF因子可能是翼状胬肉复发机制中的重要因子,自身免疫炎症...     

rno-miR-30b-5p在葡萄膜炎发病过程中对白细胞介素-10和Toll样受体4表达的调控作用

目的 探讨rno-miR-30b-5p在葡萄膜炎中对白细胞介素10(interleukin-10,IL-10)和Toll样受体4(toll-like receptor 4,TLR4)表达的调控作用.方法 构建IL-10、TLR4基因的3’端非编码区域(3'UTR)荧光素酶质粒载体以及结合位点突变质粒载体,将mo-miR-30b-5p模拟物(mimic)与构建好的报告基因载体共转染293T细胞,通过报告基因相对荧光值的表达改变检测mo-miR-30b-5p对IL-10、TLR4表达的调控作用;制备实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis,EAU)模型,通过注射视网膜光感受器间维生素A类结合蛋白乳糜液,在造模后12 d分离EAU大鼠的脾脏和淋巴结,实时荧光定量PCR检测rno-miR-30b-5p和IL-10、TLR4基因的表达情况;ELISA检测IL-10和TLR4蛋白的表达情况.结果 双荧光素酶报告基因表达结果表明,经rno-miR-30b-5p mimic干预后IL-10、TLR4野生型的报告荧光有明显的下调作用,对其预测靶位点进行突变后,突变型载体中的报告荧光也存在明显的下调作用,与阴性对照相比,差异均有统计学意义(均为P＜0.01).实时荧光定量PCR检测结果发现rno-miR-30b-5p在EAU大鼠脾脏和淋巴结中的表达均较对照组明显降低(均为P＜0.01),而IL-10 mRNA、TLR4 mRNA表达明显升高(均为P＜0.01);ELISA检测发现IL-10、TLR4蛋白表达升高,与对照组相比,差异均有统计学意义(均为P＜0.05).结论 rno-miR-30b-5p对带有IL-10、TLR4基因片段3'UTR基因表达的调控作用可能不是通过预测到的位点起作用而是通过其他的调控结合位点发挥作用;rno-miR-30b-5p在EAU大鼠脾脏和淋巴结的下调表达导致IL-10和TLR4的表达水平升高,进而调控葡萄膜炎的发展.本研究为进一步探索microRNA在葡萄膜炎发生发展进程中的调控作用、治疗葡萄膜炎奠定了基础.     

Toll样受体信号通路和糖尿病视网膜病变的关系

糖尿病视网膜病变(diabetic retinopathy,DR)是导致失明的主要疾病之一,其发病机制尚未完全阐明.Toll样受体(Toll-like recetors,TLRs)是机体免疫系统识别、感知细菌、病毒等病原体入侵的重要分子,在机体免疫防御功能中发挥着重要作用.众多的研究提示糖尿病发病与免疫系统的激活密切相关,TLRs的激活在DR的发生发展中起着重要的作用.现就Toll样受体信号通路与糖尿病视网膜病变的关系作一综述,为DR的防治提供新的治疗手段和思路.     

Toll样受体-4在内毒素诱导的葡萄膜炎中的表达及意义

目的 探讨Toll样受体-4(TLR4)在内毒素诱导的葡萄膜炎中的表达和意义.设计 实验性研究.研究对象 Wistar大鼠12只,随机分为模型组(n=6)和对照组(n=6).方法 模型组通过足底及腹腔注射霍乱弧菌内毒素诱导出葡萄膜炎,对照组注射磷酸盐缓冲液.注射后4h、10h、16h、24h裂隙灯观察眼前节反应.注射后24h通过免疫组化方法检测眼球冰冻切片及葡萄膜铺片中TLR4的表达.TLR4阳性判定标准:呈棕黄色颗粒定位于胞膜、胞浆.主要指标 眼前节炎症反应程度、葡萄膜铺片中TLR4阳性表达的细胞数量.结果 内毒素注射后4h虹膜血管扩张充血,16h前房可见纤维素渗出.模型组虹膜铺片内可见大量TLR4阳性表达细胞,位于虹膜基质层,对照组虹膜铺片内只见少量TLR4阳性表达细胞,模型组虹膜中阳性细胞数量高于对照组(P＜0.05).冰冻切片中虹膜内偶见少量TLR4表达.脉络膜和视网膜冰冻切片及铺片中均未见阳性细胞.结论 内毒素诱导的葡萄膜炎中TLR4表达增高,提示TLR4可能在急性前葡萄膜炎的发生发展中具有一定作用.(眼科,2008,17:48-51)     

内毒素诱导的大鼠葡萄膜炎的巨噬细胞中Toll样受体4的表达

目的 探讨在内毒素诱导的Wistar大鼠葡萄膜炎中Toll样受体4(TLR4)阳性细胞与虹膜组织中巨噬细胞的动态变化和分布.方法 实验研究.Wistar大鼠50只,用随机数字法随机分为5组,每组10只,分别为正常对照(0 h)组、6 h组、12 h组、24 h组及48 h组.除0 h组外其余各组均足垫部注射霍乱弧菌内毒素200μg,注射后于裂隙灯显微镜下观察双眼前节炎症反应变化.按实验分组于0、6、12、24、48 h处死大鼠.取虹膜一睫状体及脉络膜组织.通过葡萄膜铺片免疫组织化学方法检测TLR4和巨噬细胞的标记CD163的表达.人工计数虹膜中TLR4~+与CD163~+的细胞并计算细胞密度,计算圆形和多形性的CD163~+细胞占所有CD163~+细胞的百分比.进一步采用免疫荧光双标记检测TLR4和CD163共表达的情况.通过单因素方差分析分别对大鼠虹膜内阳性细胞密度以及圆形、多形性CD163~+细胞的百分比进行统计学检验.结果 正常大鼠虹膜睫状体组织不表达TLR4.6 h组有2只大鼠虹膜内可见少量TLR4~+细胞,12～48 h组所有大鼠虹膜内TLR4~+细胞明显增多(F=167.2,P＜0.001),虹膜内TLR4~+细胞密度分别为(506.1±39.5)个/mm~2(12 h组)、(492.3±54.5)个/mm~2(24 h组)及(663.8±150.2)个/mm~2 (48 h组).在注射LPS后12～48 h期间TLR4~+细胞形态无明显变化.0～48 h组大鼠虹膜内均有CD163~+细胞,0 h组圆形和多形性CD163~+细胞百分比为13%,12～48 h组其百分比约为80%,且圆形细胞主要位于虹膜基质层.免疫荧光双标记可见TLR4和CD163的共表达,TLR4位于细胞膜,CD163位于细胞质.5组大鼠脉络膜内均未见TLR4表达.结论 内毒素诱导的大鼠葡萄膜炎中虹膜内TLR4表达增高,部分虹膜固有巨噬细胞表达TLR4.TLR4可能在葡萄膜炎的发生发展中起一定作用.     

淋巴管内皮透明质酸受体-1和血管内皮生长因子-C在喉鳞癌组织中的表达

目的探讨喉鳞癌组织中淋巴管内皮透明质酸受体-1（lymphatic vessel endothelial hyaluronan receptor-1，LYVE-1）及血管内皮生长因子-C（vascular endothelial growth factor-C，VEGF-C）的表达及其与临床病理因素的关系。方法分别以酶标链霉亲和素-生物素法和Envision二步法检测40例喉鳞癌组织中LYVE-1，VEGF-C和核增殖抗原Ki67的表达，分析LYVE-1标记的淋巴管的密度、位置、增殖情况及VEGF-C表达与喉鳞癌颈淋巴结转移、T分期及肿瘤细胞分化程度等之间的关系。结果（1）颈淋巴结转移组、T3和T4组瘤巢内LYVE-1（＋）管腔密度分别高于非转移组、T1和T2组（t=4．539，P=0．000；t=3．896，P=0．000）。（2）瘤巢内LYVE-1（＋）管腔存在Ki67细胞核棕黄色着色，而瘤巢周围LYVE-1（＋）管腔未见这一现象。（3）VEGF-C在颈淋巴结转移组与无转移组、T1和T2组与T3和T4组之间的表达差异有统计学意义（χ^2=4．286，P=0．038；χ^2=4．607，P=0．032）；VEGF-C（＋）组瘤巢内LYVE-1（＋）管腔密度高于VEGF-C（-）组，差异有统计学意义（t=2．128，P=0．044）。结论喉鳞癌组织中LYVE-1标记的肿瘤淋巴管生成与肿瘤浸润、淋巴结转移、VEGF-C表达有关，淋巴管生成及VEGF-C表达在喉癌的发展过程中发挥重要的作用。     

喉鳞癌中扭曲基因、上皮型钙黏附蛋白和神经型钙黏附蛋白基因表达及相关研究简

目的 研究转录因子扭曲基因（Twist）在喉鳞癌（LSCC）中的表达及其通过对上皮型钙黏附蛋白（E-cadherin）、神经型钙黏附蛋白（N-cadherin）的作用在判断肿瘤的生物学行为方面的意义。方法 采用免疫组织化学SP法及反转录聚合酶链反应（RT-PCR）方法检测49例喉鳞癌及20例癌旁组织中Twist、E-cadherin和N-cadherin基因的表达情况。结果 Twist在喉癌组织中的阳性表达率（61.22%）明显高于癌旁组织（25%）（P〈0.01）;Twist在喉癌组织中的mRNA相对表达量（0.93±0.39）明显高于癌旁组织（0.57±0.24）（P〈0.01）;Twist在喉癌中蛋白及mRNA表达均与淋巴结转移、临床分期相关,而与肿瘤病理学分级、临床分型及年龄无关。在mRNA及蛋白水平,Twist与E-cadherin,N-cadherin与E-cadherin的表达均呈负相关;Twist与N-cadherin均呈正相关。结论 Twist在喉鳞癌中过度表达, 可能通过分别上调N-cadherin和下调E-cadherin的表达,进而在喉鳞癌的浸润、转移过程中发挥重要作用。     

眼睑鳞状细胞癌移植瘤模型建立及其VEGF的表达研究

目的建立眼睑鳞状细胞癌(SCC)移植瘤模型并研究其血管内皮生长因子(VEGF)的表达。方法BALB/c裸鼠40只,随机平均分为实验组和对照组,实验组裸鼠右上睑皮下接种Tca8113细胞建立眼睑SCC移植瘤,接种后第15、30 d,应用酶联免疫吸附检测(ELISA)法检测所有裸鼠血清中VEGF蛋白水平,并采用免疫组织化学法检测实验组移植瘤组织和对照组正常眼睑组织中VEGF的表达。结果实验组裸鼠右上睑均有SCC移植瘤形成,接种后第15、30 d,实验组裸鼠血清中VEGF质量浓度(188.6 pg/m l±79.93 pg/m l和200 pg/m l±75.28 pg/m l)均显著高于对照组(94.8 pg/m l±31.41 pg/m l和90.4 pg/m l±28.62 pg/m l)(P<0.01),两组间VEGF染色分数经统计学分析也存在显著差异(2.6±0.16和2.38±0.19,0.64±0.18和0.52±0.15)(P<0.01)。结论Tca8113细胞裸鼠眼睑皮下接种是建立眼睑SCC移植瘤的可行方法,靶向VEGF的基因治疗可望为眼睑SCC治疗提供新的途径。     

Periocular squamous cell carcinoma

Squamous cell carcinoma (SCC) is the second most common eyelid malignancy and its incidence is increasing. Because of its variable clinical presentation, SCC may be difficult for even the experienced clinician to diagnose. Hence, all suspicious lesions warrant biopsy. As SCC behaviour may range widely in aggression, management should be individualized based on tumour (e.g. size, location, grade, histological subtype, previous recurrence, perineural invasion) and patient factors (e.g. age, health). Treatment of eyelid SCC should include margin control whenever possible for the highest cure rate in this high-risk area. Immediate histological monitoring of surgical margins with frozen sections or Mohs' micrographic surgery also allows for smaller margins of excision in an area where tissue conservation is important. Other special considerations in the periocular area include maintaining a high level of suspicion for perineural invasion as this may be associated with a poorer prognosis.     

Conjunctival squamous cell carcinoma in Tanzania

AIMS: To assess changes in incidence of conjunctival squamous cell carcinoma over a 22 year period in Tanzania and to analyse possible reasons for change. METHODS: Retrospective analysis of records from a Tanzanian pathology department serving north and central Tanzania from 1976 to 1997; medical record analysis of cases of conjunctival squamous cell carcinoma presenting in the last 2 years of the study. RESULTS: There was a sharp rise in the incidence of conjunctival squamous cell carcinoma in the last 3 years of the study (1995-7). The mean age of patients presenting with the condition over the full period was 44.7 years (95% confidence interval 42.4-46.9 years). In the final 2 years of the study the mean length of history on presentation was 3.1 months (2.1-4.0 months). Several patients had a previous history of chronic conjunctival disease such as allergic conjunctivitis and trachoma; one had had a conjunctival papilloma excised previously. Only five patients had been tested for HIV status, but of these four were positive. CONCLUSION: Tanzania is experiencing an epidemic of conjunctival squamous cell carcinoma similar to that seen in other African countries. Often the tumours are aggressive and occur in patients of relatively young age. The epidemic appears to be related to HIV infection, on a background of ultraviolet light exposure. Previous chronic conjunctival disease and exposure to human papillomavirus may also have a role.     

Expression of p63 in conjunctival intraepithelial neoplasia and squamous cell carcinoma

Background p63 is a homologue of the tumour suppressor gene p53, which is expressed in human basal squamous epithelium. Some investigators maintain that p63 plays a role in the development of squamous epithelium and, despite its homology to p53, it is considered to act as an oncogene. This study investigated the expression of p63 in conjunctival intraepithelial neoplasia of different grades, and conjunctival squamous cell carcinoma and its correlation to the proliferation marker MIB-1. Material and methods Seventeen conjunctival specimens excised with the suspicion of either conjunctival intraepithelial neoplasia (CIN) or squamous cell carcinoma were diagnosed histologically as follows: 2 squamous cell carcinomas of the conjunctiva, 2 CIN grade I, 3 CIN grade II, 7 CIN grade III, 2 CIN with beginning invasion and 1 normal conjunctiva with no dysplasia. Sixteen microscopically-normal postmortem conjunctival specimens and normal conjunctiva, CIN and carcinoma specimens were stained immunohistochemically with antibodies against p63 and MIB-1. At least 500 cells per specimen were counted and the percentage of positively-stained cells of each antibody was calculated. Results A mean of 80% (57–89%) of the dysplastic cells from the CIN specimens stained positively with antibodies against p63, especially in the lower two-thirds of the epithelium, statistically significantly more compared with the normal specimens (9–55%, mean 36%, p<0.001). Nevertheless, we did not find a correlation between the percentage of p63-positive cells and the differentiation grade of the malignant specimens. MIB-1 positivity was shown by 0–1% of the cells in the normal postmortem controls, by 3–30% (mean 12%) of the cells in the basal and occasionally in the middle layer of the CIN specimens, and 16–61% (mean 23%) in the carcinoma specimens. Conclusion In conjunctival intraepithelial neoplasia and squamous cell carcinoma of the conjunctiva, p63 is preferentially expressed in the immature dysplastic epithelial cells. Its staining does not correlate with MIB-1-expression, and therefore does not appear to be linked to cell proliferation.     

Periorbital desmoplastic squamous cell carcinoma

Desmoplasia is the formation of a dense collagenous stroma around a neoplasm. It occurs in a variety of malignancies including squamous cell carcinoma (SCC). While desmoplasia is uncommonly seen in cutaneous SCC, it is an independent risk factor for recurrence and metastasis. We report a case series of desmoplastic SCC in the periorbital region. Seven cases were identified: the median age was 68, four were men. The mean follow-up was 48 months. Two patients (29%) had aggressive local recurrence: one required salvage surgery including orbital exenteration, parotidectomy, and neck dissection to excise involved parotid and cervical lymph nodes; the other required repeat excision and adjuvant radiotherapy. Desmoplastic SCC is an uncommon but highly aggressive subtype. In the periorbital region, due to the high risk of orbital invasion, it is potentially sight and life-threatening.     

Orbital squamous cell carcinoma following retinal detachment surgery

Background: Orbital squamous cell carcinoma following retinal detachment surgery is a rare condition. A proposed pathogenesis involves implantation of conjunctival squamous epithelium at the time of surgery, with subsequent malignant transformation. Methods/Results: An 81 -year-old Caucasian man presented with a six-month history of a discharging painful red right eye. Ten years earlier a right scleral buckling with an encircling sponge exoplant had been performed. Examination revealed exposure of the sponge, and a large mass in the superomedial conjunctival fornix. Computed tomography revealed a mass invading around the globe in the region of the exoplant. Biopsy revealed squamous cell carcinoma. Orbital exenteration was performed. Conclusion: Orbital squamous cell carcinoma may arise following retinal detachment surgery presumably due to iatrogenic conjunctival implantation deep in the orbit.     

Conjunctival squamous cell carcinoma: atypical presentation of HIV

Metastatic squamous cell carcinoma (SCC) of the conjunctiva is uncommon in Western countries. A Nigerian patient who presented with right lower lid swelling treated as a chalazion is described here. She re-presented 5 months later with widespread metastatic conjunctival SCC and positive HIV serology. Conjunctival SCC may be difficult to distinguish on clinical appearance alone and in an era of increasing global travel, young patients with a history of high ultraviolet-B radiation exposure and from countries with a high HIV prevalence should be given a low threshold for excision biopsy.