龙胆泻肝汤抑制葡萄膜炎大鼠Notch信号通路活化的作用

目的　探讨龙胆泻肝汤（Longdan Xiegan Decoction，LXD）对实验性自身免疫性葡萄膜炎（experimental autoimmune uveitis，EAU）大鼠Notch信号通路活化的抑制作用及其对辅助性T细胞17（T helper 17，Th17）和调节性T细胞（regulatory T cell，Treg）表达水平的影响。方法　随机将雌性Lewis大鼠分为正常对照（NC）组、EAU模型组、LXD干预组。EAU模型组和LXD干预组大鼠诱导EAU，免疫后LXD干预组大鼠每天给予LXD灌胃处理，EAU模型组大鼠给予等量生理盐水灌胃。免疫后12 d观察大鼠眼部炎症表现，取三组大鼠眼球进行病理切片，观察病理学变化；实时荧光定量PCR（quantitative polymerase chain reaction，QT-PCR）和酶联免疫吸附实验（enzyme-linked immunosorbent assay，ELISA）检测免疫后12 d三组大鼠脾脏、淋巴结及眼组织中Notch1、DLL4、白细胞介素10（interleukin 10,IL-10）和IL-17 mRNA及蛋白的表达；流式细胞仪检测三组大鼠各组织中Th17、Treg细胞的表达。结果　病理检查结果表明，LXD对EAU大鼠眼部组织结构有明显的保护作用。QT-PCR和ELISA检测结果发现，与NC组相比，LXD干预组大鼠脾脏、淋巴结和眼组织中Notch1、DLL4、IL-10、IL-17 mRNA和蛋白表达水平均升高，但除IL-10外，其他明显低于EAU模型组（均为P<0.05）；流式细胞仪检测结果发现，EAU模型组大鼠的各组织中Th17/Treg比例均高于NC组，经LXD干预后，Th17细胞表达水平下降，Treg表达水平升高，两者比例趋向平衡。结论　LXD可有效降低EAU大鼠脾脏、淋巴结、眼组织中Notch1、DLL4、IL-10和IL-17 mRNA和蛋白的表达水平，改善Th17/Treg细胞比例的平衡，从而有效减轻EAU大鼠的眼部炎症，保护眼部组织结构，调节全身及眼部的免疫状态。     

间充质干细胞对大鼠实验性自身免疫性葡萄膜炎视网膜中白细胞介素17表达的影响

背景 研究显示Th17细胞是实验性自身免疫性葡萄膜炎(EAU)发病的重要致炎细胞群,而白细胞介素17( IL-17)作为Th17细胞的标志性因子参与了EAU的发生与发展.间充质干细胞(MSCs)作为一种免疫调节剂在多种自身免疫性疾病中对Th17具有抑制作用. 目的 研究MSCs对EAU的治疗作用以及对IL-17在大鼠视网膜表达的影响.方法 从10只SPF级Wistar大鼠股骨骨髓中分离、培养MSCs并进行传代,第3～5代细胞用于实验.54只SPF级6～8周龄Lewis大鼠按随机数字表法分为实验组48只和正常对照组6只.光感受器间维牛素A类结合蛋白(IRBP)与含有结核杆菌素HR37a的2.5 g/L弗氏完全佐剂(CFA)等体积混合后行实验组大鼠单后足垫皮下注射建立EAU动物模型,然后按照分层随机原则分为模型对照组和MSCs治疗组,每组24只大鼠.MSCs治疗组于造模的同时行1 ml MSCs尾静脉注射(5×106个／ml),连续注射3d,模型对照组以同法注射等体积PBS.注射后每日在裂隙灯显微镜下观察大鼠眼部炎症反应,并根据Caspi临床分级进行评分.分别于造模后9、12、15、20 d随机选取模型对照组和MSCs治疗组各6只大鼠制备视网膜切片,采用组织病理学方法观察大鼠视网膜形态学改变和炎症反应,参照Caspi组织学分级法进行评分.采用免疫组织化学染色法检测造模后9、12、15、20d大鼠视网膜中IL-17的表达.结果 培养的细胞呈梭形生长,漩涡状排列,经流式细胞术鉴定CD29、CD44表达阳性,CD45、CD34表达阴性.MSCs治疗组造模后9、12、15、20 d眼前节炎症反应评分均低于模型对照组(U=2.815,P=0.005;U=2.768,P=0.006;U=2.900,P=0.004;U=2.855,P=0.004),视网膜组织学检测炎症评分均低于模型对照组,差异均有统计学意义( U=2.345,P=0.019:U=2.559,P=0.011;U=2.166,P=0.030;U=2.373,P=0.018).免疫组织化学染色显示,MSCs治疗组造模后9、12、15、20d大鼠视网膜IL-17蛋白阳性表达的平均吸光度(A)值分别为26.47±5.68、77.78±9.65、47.02±6.68和26.59±5.94,明显低于模型对照组的45.34±4.63、105.95±10.74、64.11±9.76和37.02±6.51,差异均有统计学意义(t=6.305,P=0.000:t=4.799,P=0.001:t=3.540,P=0.005;t=2.900,P=0.016).结论 MSCs能减轻EAU的程度并抑制EAU的进展,降低了1L-17在眼组织中的表达.     

CD4＋CD25＋T细胞在实验性自身免疫性葡萄膜视网膜炎中的作用

背景实验性自身免疫性葡萄膜视网膜炎（EAU）已被证明是一种由T淋巴细胞介导的器官特异性自限性疾病。研究表明 CD4＋CD25＋T细胞可能参与了EAu的调控,但其作用机制尚有待研究。目的探讨EAU中 CD4＋CD25＋T细胞的表达变化。方法按照Caspi的方法提纯牛视网膜S抗原,与弗氏完全佐剂混合后,于24只Lewis大鼠右后足底部注射0．1ml制作EAU模型,4只未处理大鼠作为正常对照组。免疫后每日州裂隙灯显微镜观察大鼠眼部变化。造模后7、12、15、21d处死动物,取大鼠视网膜、引流淋巴结、脾脏,对大鼠眼组织切片进行苏木精一伊红染色前进行病理评分。对各时间点收集的大鼠视网膜、引流淋巴结、脾脏分别制备单细胞悬液,流式细胞仪检测各时间点3种组织中 CD4＋CD25＋T细胞的表达情7兄。结果造模7d后大鼠睫状充血,虹膜血管扩张;15d后炎症达高峰,前房大量炎性渗出;21d后炎症消退。眼部病理评分结果与临床所见一致,造模15d组病理评分与其他各组比较差异均有统计学意义（P=0．000）。正常对照组大鼠脾脏和淋巴结中有2．0％CD4＋T细胞表达CD25,造模组 CD4＋CD25＋T细胞表达增加,亓在EAU高峰期达最高,EAU消退期轻微下降,与正常对照组比较差异有统计学意义（P=0．000）。结论 CD4＋CD25＋T细胞在EAU动物模型炎症组织中表达的动态变化与炎症反应有关,提示 CD4＋CD25＋T细胞参与EAU的发生发展和消退过程。     

清开灵眼用凝胶对大鼠实验性自身免疫性葡萄膜炎的治疗作用及其机制

背景葡萄膜炎是常见的致盲眼病,多反复发作,目前主要的治疗方法是应用糖皮质激素和免疫抑制剂,但不良反应较多,寻求安全、有效的治疗方法至关重要。目的观察清开灵眼用凝胶对大鼠实验性自身免疫性葡萄膜炎（EAU）的疗效,并探讨其作用机制。方法SPF级雌性Lewis大鼠27只,皮下注射200斗l含100μg光感受器间维生素A类结合蛋白（IRBP）1177—1191多肽、100μl完全弗氏佐剂（CFA）、100μg结核菌素及100μlPBS的乳化液,在大鼠两足垫处、尾根部两侧及脊背正中均匀注射5个点建立EAU动物模型。将免疫后的大鼠按随机数字表法随机分为模型对照组、清开灵眼用凝胶组和妥布霉素地塞米松组。大鼠免疫后第7天开始点眼,模型对照组用生理盐水点眼,清开灵眼用凝胶组和妥布霉素地塞米松组分别用相应的药物点眼,每日3次,连续用药7d。从免疫后第1天开始裂隙灯显微镜下观察大鼠眼前节炎症反应并进行炎症评分;免疫后第14天过量麻醉法处死实验动物并获取眼球标本,采用组织病理学方法观察大鼠前房、虹膜和睫状体的炎性细胞浸润情况;采用流式细胞仪测定大鼠脾脏、淋巴结分离的T细胞悬液中CD4^＋和CD8^＋细胞百分比、CD4^＋／CD8^＋值以及Th1细胞和Th17细胞的比例。结果模型对照组大鼠免疫后第9天开始出现眼部炎症表现,第11天炎症反应达高峰期,而清开灵眼用凝胶组和妥布霉素地塞米松组大鼠眼部炎症反应的发生较模型对照组晚,炎症反应轻,病程短。免疫后13d,3个组大鼠眼部炎症评分的总体差异有统计学意义（F=26．52,P=0．00）。清开灵眼用凝胶组和妥布霉素地塞米松组的炎症评分明显低于模型对照组,差异均有统计学意义（t=6．72、10．11,P〈0．05）。组织病理学检查发现,模型对照组大鼠前房、虹膜及睫状体组织中可见炎性细?     

间充质干细胞对兔自身免疫性干眼模型病理改变及细胞因子表达的影响

目的 建立兔自身免疫性干眼模型,观察间充质干细胞(mesenchymal stem cells,MSCs)治疗后兔自身免疫性干眼的组织病理学变化及泪腺组织中细胞因子表达的变化.方法 取健康成年雌性新西兰白兔24只24眼,采用随机数学表法将兔分为正常对照组、干眼模型组和MSCs治疗组,每组各8只8眼.MSCs治疗6周后,观察兔眼组织病理学改变,实时荧光定量PCR检测泪腺组织中Th1、Th17细胞分化相关细胞因子mRNA相对表达量.流式细胞仪检测干眼模型组和MSCs治疗组兔泪腺组织和脾脏组织中调节性T细胞(CD4+ Foxp3+细胞)占淋巴细胞的比例.结果 MSCs治疗6周后,取各组兔泪腺并行HE染色显示:正常对照组未见或仅见少量淋巴细胞浸润;干眼模型组泪腺内部分腺体细胞出现萎缩,腺管周围和小血管周围可见散在分布的淋巴细胞汇集区;与干眼模型组相比,MSCs治疗组泪腺内淋巴细胞浸润减轻,腺泡细胞形态较好.与干眼模型组比较,MSCs治疗组结膜组织中淋巴细胞浸润聚集显著减轻,上皮结构完整,变性和萎缩细胞较少见.MSCs治疗组泪腺组织中Th1特征性细胞因子IFN-γ以及转录因子T-bet mRNA表达水平较干眼模型组明显下降,差异均有统计学意义(均为P ＜0.05);Th17细胞分化相关细胞因子IL-17表达水平有下降趋势,但两组差异无统计学意义(P＞0.05);其转录因子RORa mRNA的表达水平较干眼模型组显著降低,差异有统计学意义(P＜0.05).MSCs治疗组泪腺组织中炎症因子TNF-α表达较干眼模型组显著降低,而抑炎因子TGF-β的表达较干眼模型组显著升高,差异均有统计学意义(均为P＜0.05).泪腺组织中干眼模型组调节性T细胞(CD4+ Foxp3+细胞)占淋巴细胞的比例为10.0％,而MSCs治疗组中调节性T细胞占淋巴细胞的比例为27.8％,MSCs治疗组调节性T细胞较干眼模型组显著升高,差异有统计学意义(P＜0.05).结论 MSCs可以减轻自身免疫性干眼的组织病理学改变,对自身免疫性干眼具有一定的免疫调节及治疗作用,其免疫调控作用可能与调节抑炎因子和促炎因子平衡有关.     

龙胆泻肝汤对葡萄膜炎大鼠Notch信号通路的干预作用

目的　探讨龙胆泻肝汤对实验性自身免疫性葡萄膜炎（experimental autoimmune uveitis,EAU）大鼠Notch信号通路相关基因表达的影响。方法　Lewis大鼠用随机数字表法分为正常对照组、EAU模型组和龙胆泻肝汤干预组,后两组诱导EAU模型,龙胆泻肝汤干预组造模后用龙胆泻肝汤灌胃。免疫后12 d分别分离三组大鼠的脾脏和淋巴结收集CD4⁺T细胞,流式细胞仪检测Th17、Treg细胞的表达水平;实时荧光定量PCR（qRT-PCR）检测Notch1、Notch2、Notch3、Notch4基因的表达情况;ELISA检测Notch信号通路相关蛋白的表达水平。结果　流式细胞仪检测发现,与正常对照组相比,EAU模型组大鼠脾脏和淋巴结中Th17细胞水平明显升高,Treg细胞水平降低;与EAU模型组相比,龙胆泻肝汤干预组大鼠的Th17细胞水平明显下降,Treg细胞水平升高,两者比例逐渐恢复均衡;qRT-PCR检测发现龙胆泻肝汤干预组大鼠脾脏和淋巴结中Notch1、Notch2和Notch4基因的表达在免疫后12 d高于正常对照组（均为P<0.01）,但显著低于EAU模型组（均为P<0.01）,Notch3基因在大鼠脾脏和淋巴结未检测到表达;ELISA检测结果发现,龙胆泻肝汤干预组大鼠脾脏和淋巴结中Notch1、Notch2和Notch4蛋白表达水平在免疫后12 d虽然高于正常对照组（均为P<0.01）,但显著低于EAU模型组大鼠（均为P<0.01）。结论　龙胆泻肝汤可有效缓解EAU大鼠中Th17/Treg比例失衡状态,其作用机制可能涉及到Notch信号通路调节的nave CD4⁺T细胞向Th17 和Treg细胞的分化。     

调节性T细胞在实验性自身免疫性葡萄膜炎中的作用

目的探讨CD4^＋调节性T细胞（Treg）在实验性自身免疫性葡萄膜炎（EAU）病程进展中的作用。方法通过IRBP161-180和完全弗氏佐剂皮下免疫,在B10RⅢ小鼠诱导产生EAU,对眼内炎症行临床检查并对其严重程度予以评分。通过免疫磁珠分选EAU恢复期小鼠脾脏CD4^＋T细胞、CD8^＋T细胞、CD4^＋CD25^＋T细胞和CD4^＋CD25^-T细胞。通过流式细胞术分析脾脏CD4^＋CD25^＋Foxp3^＋T细胞的比例。通过体内过继转移评价Treg的抑制效应。结果小鼠诱导EAU后产生眼内炎症反应。将EAU恢复期小鼠脾细胞、脾CD4＋T细胞或脾CD4^＋CD25^＋T细胞过继转移入正常小鼠继而皮下免疫诱导EAU,眼底检查显示受者小鼠眼内炎症程度明显降低。相反,正常小鼠脾细胞、EAU恢复期脾CD8^＋T细胞或CD4^＋CD25^-T细胞过继转移无此效应。流式细胞分析显示随着EAU的发展,脾内CD4^＋CD25^＋T细胞比例和CD4^＋CD25^＋Foxp3^＋T细胞比例逐步升高,并在恢复期维持较高水平。结论 CD4^＋Treg随着EAU的病程进展数量增多,它们可能参与EAU的自发缓解。     

Treg细胞在实验性自身免疫性葡萄膜炎发病过程中的动态变化

背景 研究表明,调节性T细胞(Treg)是一类负向调控免疫应答的T细胞亚群,在维持免疫稳态和免疫耐受方面发挥重要作用.自身免疫性葡萄膜炎是一种免疫性眼病,Treg细胞在自身免疫性葡萄膜炎发生和发展过程中的调控作用尚不完全清楚. 目的 观察Treg细胞在实验性自身免疫性葡萄膜炎(EAU)大鼠发病过程中的动态变化.方法 选取84只6～8周龄SPF级Lewis大鼠,采用随机数字表法随机分为模型组和对照组.模型组于大鼠双后足垫、腹部双侧及背部皮下注射光感受器间维生素A类结合蛋白(IRBP)1177-1191、结核菌素(TB)、完全弗氏佐剂(CFA)和PBS混合乳化剂共300μl,对照组大鼠以同样方法皮下注射等容量不含IRBP的TB与CFA乳化剂.分别于造模后第9、13、18、23、28、35、48天观察模型组和对照组大鼠的眼部炎症症状并根据严重程度进行炎症评分.于造模后上述时间点分别处死模型组及对照组大鼠各6只,采用常规组织病理学方法观察各组大鼠眼部虹膜、睫状体和视网膜组织形态变化;分离大鼠脾脏淋巴细胞,采用流式细胞术检测大鼠脾脏悬液中Treg细胞特异性标志物Foxp3标记细胞比例;采用实时荧光定量PCR法检测大鼠脾脏淋巴细胞中Foxp3 mRNA相对表达量变化. 结果 免疫后第8天模型组大鼠虹膜血管扩张充血,开始出现眼部炎症表现,免疫后第13天虹膜血管明显扩张,前房可见渗出和积脓,瞳孔区有膜样渗出,炎症评分最高,为(3.75±0.42)分,之后眼部炎症反应逐渐减轻,至免疫后第23天炎症反应接近消失,造模后第7、11、13、15、17、19、21天间模型鼠眼炎症反应评分总体比较差异有统计学意义(F=81.709,P＜0.001).对照组大鼠眼部检查正常.组织病理学观察发现,模型组大鼠虹膜、睫状体、视网膜等组织有中性粒细胞、淋巴细胞和单核细胞浸润,组织结构排列疏松,以免疫后第13天最为明显,此后逐渐减轻,至免疫后第23天虹膜、睫状体和视网膜组织结构接近正常,炎性细胞浸润消失.流式细胞技术检测发现,免疫后第13、18、23、28、35、48天模型组大鼠脾脏Foxp3标记细胞比例分别为(5.50±0.64)％、(13.36±0.98)％、(10.34±0.79)％、(9.58±1.02)、(6.73±0.81)％和(5.58±0.47)％,明显高于对照组的(2.80±0.38)％、(3.36±0.53)％、(3.65±0.57)％、(3.37±0.43)％、(3.33±0.50)％和(3.13±0.61)％,差异均有统计学意义(t=-6.272、-15.556、-11.910、-9.753、-6.154、-5.491,均P＜0.01).模型组和对照组造模后各时间点大鼠脾脏淋巴细胞中Foxp3 mRNA的相对表达量变化与Foxp3标记细胞比例变化趋势基本一致. 结论 EAU大鼠葡萄膜炎的发病及转归与Treg细胞数量和功能变化密切相关.     

龙胆泻肝汤对实验性自身免疫性葡萄膜炎大鼠补体C4、MBL2表达的影响

目的　探讨龙胆泻肝汤（Longdan Xiegan decoction,LXD）对实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis,EAU)大鼠的治疗作用及对血清中C4、MBL2蛋白表达水平的影响。方法　54只Lewis大鼠用随机数字表法分为正常对照组、EAU组和LXD组,其中EAU组、LXD组大鼠制备EAU模型,LXD组造模后使用LXD每天灌胃处理,EAU组给予等量生理盐水灌胃。免疫后12 d使用激光扫描检眼镜（scanning laser ophthalmoscope,SLO）观察三组大鼠眼底炎症,并取三组大鼠同侧眼球进行病理切片,观察视网膜组织病理学变化;分离三组大鼠的脾脏和淋巴结,收集T淋巴细胞,流式细胞仪检测CD4⁺/CD8⁺细胞比例的变化;酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）检测血清中C4、MBL2蛋白的表达水平。结果　SLO检查结果显示,与正常对照组相比,EAU组大鼠屈光间质不清,无法观察眼底视网膜及血管情况;LXD组大鼠眼底血管迂曲扩张,屈光间质混浊,视盘边界模糊不清,但较EAU组大鼠症状轻。组织病理学检查结果显示,与正常对照组相比,EAU组大鼠眼组织结构紊乱,视网膜全层破坏,视网膜内可见大量炎性细胞浸润;LXD组大鼠视网膜仅表现为轻、中度炎性细胞浸润。流式细胞仪检测结果发现,EAU组大鼠脾脏、淋巴结中CD4⁺/CD8⁺比值均高于正常对照组;LXD组大鼠的CD4⁺ T细胞表达水平下降,CD8⁺ T细胞表达水平升高,两者比例趋于平衡。ELISA检测结果发现,与正常对照组相比,EAU组大鼠免疫后12 d、16 d、20 d血清C4蛋白水平均显著升高,与EAU组相比,LXD组免疫后12 d、16 d、20 d血清C4蛋白水平均明显降低,差异均有统计学意义（均为P<0.05）;同时,EAU组各时间点血清MBL2蛋白水平明显降低,而LXD组较EAU组各时间点血清MBL2蛋白水平均显著升高,差异均有统计学意义（均为P<0.01）。结论　LXD可有效缓解EAU大鼠眼内炎症,改善脾脏、淋巴结中CD4⁺/CD8⁺细胞比例失衡,同时降低血清中补体C4蛋白表达水平,上调MBL2蛋白表达水平,促进补体系统恢复平衡,加快葡萄膜炎的炎症消退,从而达到治疗EAU的作用。     

实验性自身免疫性葡萄膜炎小鼠T细胞变化的动态研究

目的：了解实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis, EAU)不同进展阶段小鼠T细胞动态变化,对葡萄膜炎治疗方案的优化及疗效评价提供指导。

方法：用CFA+PTX+IRBP对6～10周龄雌性C57BL/6小鼠后肢及尾部皮下进行三点免疫建立EAU模型,于免疫3,7,14,21,28d取外周血行流式细胞术检测。

结果：用特异性抗原IRBP免疫后,EAU疾病在第14d左右产生,在第21d达最高峰,以后开始逐渐缓解。随着EAU疾病的发生,CD4⁺CD25⁺调节性T细胞、CD4⁺CD3⁺辅助T细胞数量均有增加,CD4⁺CD25⁺调节性T细胞增加更为明显,CD4⁺CD25⁺Foxp3⁺调节性T细胞数量第21d达到高峰,第28d开始下降,CD4⁺CD25⁺Foxp3⁺/CD4⁺CD25^-Foxp3⁺比值从第3d开始逐渐增加,到第21d达到高峰,从第28d开始下降。

结论：EAU疾病的发生和转归与CD4⁺CD25^+/-Foxp3⁺Treg细胞密切相关,CD4⁺CD25^+/-Foxp3⁺Treg细胞为阐明EAU的缓解机制、预防和治疗人类葡萄膜炎提供了新思路。     

Influence of endothelial cell count of donor's cornea on endothelial cell loss

In an experimental study porcine corneas were trephined using different trephining procedures. For every series of trephinations, the initial cell count of the donor cornea was found to play a role in the resulting damage induced by the cutting procedure. Pretreatment of corneas and modifications of trephining conditions did not change the negative correlations between cell count and damage due to trephining.     

Age-dependent susceptibility of the retinal ganglion cell layer to cell death

PURPOSE: The purpose of this study was to determine the susceptibility of the retinal ganglion cell layer (GCL) to apoptosis after optic nerve transection and excitotoxic stimulus and to investigate the regulation of apoptosis in the GCL during development. The authors also sought to determine the role played by caspases in cell death and their expression during development. METHODS: TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis during mouse retinal development from postnatal day (P)3 to P5 and in retinal explant sections under various conditions. The expression of active caspases was determined by immunohistochemistry (IHC) using an antibody that detects the cleaved large subunit. IHC was also used to detect the expression levels of procaspase-3, procaspase-9, and Apaf-1 in P6 and P60 whole eye sections. Retinal ganglion cells at ages P6 and P60 were purified by immunopanning, total RNA was extracted, and mRNA levels of the above proteins were determined by semiquantitative PCR. RESULTS: After optic nerve transection, a significant number of TUNEL-positive cells were seen 24 hours after lesion in P6 retinas. This death was caspase dependent, as shown by IHC and caspase inhibition with zVAD-fmk. In contrast, adult GCL was resistant to apoptosis under these conditions. Similarly, after excitotoxic stimulus, the GCL of the P6 retinas underwent apoptosis at 6 hours and was caspase dependent, whereas adult GCL was resistant. Developmental apoptosis in the GCL between P2 and P6 was shown to involve caspase-3 and caspase-9. Significant downregulation of Apaf-1 and caspase-3 was detected in the P60 GCL at both the mRNA and the protein levels. CONCLUSIONS: Adult GCL is more resistant to apoptosis than neonatal GCL after ON transection and excitotoxic stimulus. The expression of caspase-3 and Apaf-1 is significantly reduced in adult GCL. The authors suggest that age-dependent susceptibility to apoptosis may be caused by this reduced expression.     

角膜缘干细胞培养上清液对血管内皮细胞增殖的影响

目的：观察角膜缘干细胞培养液对血管内皮细胞增殖的影响。

方法：分别培养角膜缘干细胞及球结膜上皮细胞,并通过免疫组化检测AE-5蛋白鉴别角膜缘干细胞。搜集两组培养细胞的上清液加入培养的人脐静脉血管内皮细胞中,并设立对照组。培养24h后通过MTT法检测细胞增殖情况并进行统计学分析。

结果：角膜缘干细胞AE-5染色呈阴性,而球结膜上皮细胞为阳性。加入角膜缘干细球结膜上皮细胞和对照组培养液的血管内皮细胞MTT值分别为2.097±0.079,1.981±0.034和1.990±0.044。三组间有显著性差异(F=9.169,P=0.000)。加入角膜缘干细胞培养上清液的血管内皮细胞增殖活性明显高于其他两组(P=0.005和P=0.007)。

结论：角膜缘干细胞培养液能够促进血管内皮细胞的增殖,这可能是角膜缘干细胞的特征。本研究从功能学角度为角膜缘干细胞理论提供更多的依据。     

Differential cell adhesion and expression of N-cadherin among retinoblastoma cell lines.

Retinoblastoma (RB) is the most common intraocular childhood cancer, but little is known regarding its cellular biology and the factors that might regulate its progression and metastatic potential. Although alterations in cell-adhesion interactions could contribute to the metastatic behavior of RB, no specific cell-adhesion molecules have been identified in RB cells. The current study examined the adhesive properties of three RB cell lines (Weri-Rb1, Weri-Rb27, and Y79) and determined whether N-cadherin, a specific cell-adhesion protein expressed during normal retinal development, was expressed by these cell lines. This study revealed marked differences in the cell-adhesive properties of the three cell lines with respect to several parameters assayed, including cell morphology, calcium dependence of the cell adhesion, and the presence of N-cadherin. N-cadherin was expressed by human RB cells, but the level of expression, determined both functionally and biochemically, varied among different RB cell lines.     

Antioxidant effects of mast cell inhibitors in a human conjunctival cell line

PURPOSE: The aim of this study was to evaluate in vitro antioxidant effects of two mast cells inhibitors. METHODS: Cytotoxicity tests were done on a continuous human conjunctival cell line using microplate cold light cytofluorimetry. Membrane integrity (neutral red test), DNA condensation (Hoechst 33342 test), and reactive oxygen species (ROS) production (dichlorofluoresceine diacetate and hydroethidine tests) were evaluated on living cells treated with sodium cromoglycate and N-acetyl-aspartyl glutamic acid (NAAGA) preserved (benzalkonium chloride: BAC at 0.01%) and unpreserved after 60 minutes of treatment or 60 minutes and 24 hours of cell recovery. They were tested pure and at a 1/10 dilution. ROS production was also evaluated after a 60 minute pretreatment with antiallergic drugs and a 15-minute treatment with BAC, according to previous experiments performed on BAC showing its ROS production properties. RESULTS: No cytotoxicity was observed with the unpreserved formulations of antiallergic drugs. An apoptotic phenomenom was suggested with preserved drugs after a 1-hour treatment, whereas a necrotic mechanism appeared after a 24-hour cell recovery period. A ROS production decrease was observed with the two preserved and unpreserved drugs tested (p<0.001 compared to BAC) even if it was significantly higher with cromoglycate formulations. A ROS production decrease also was detected after a pretreatment with antiallergic drugs and treatment with BAC (p<0.001 compared to BAC alone). CONCLUSION: In vitro, no cytotoxicity was found with the two unpreserved mast cell inhibitors tested. An antioxidant effect also was observed with these two molecules; sodium cromoglycate appeared to be the best free radical scavenger.     

Protein kinase C regulation of corneal endothelial cell proliferation and cell cycle

PURPOSE: The purpose of this study was to determine the role of protein kinase C (PKC) in corneal endothelial cell proliferation. METHODS: Immunocytochemistry and Western blotting were used to define the PKC isoforms expressed in primary cultures of rat corneal endothelial cells. For proliferation studies, primary cultures of rat corneal endothelial cells were serum-starved for 48 hours and incubated for 2 hours with the PKC inhibitors staurosporine (10(-9) to 10(-7) M), chelerythrine (10(-9) to 5 x 10(-8) M), or calphostin C (10(-9) to 10(-7) M). Individual PKC isoforms were inhibited using PKCalpha antisense oligonucleotide transfection or exposure for 1 hour to myristoylated, pseudosubstrate-derived peptide inhibitors against PKCalpha, -alphassgamma, -epsilon, and -delta (10(-8) to 10(-6) M). Cells were then stimulated with 2.5% serum for 24 hours. Cell proliferation was measured with bromodeoxyuridine (BrDU) and Ki67 immunocytochemistry. Protein level of cyclin E was determined by Western blotting. RESULTS: PKCalpha, -ssII, -delta, -epsilon, -iota, -eta, -gamma, and -theta were detected in corneal endothelial cells. Maximum inhibition of PKC with staurosporine, calphostin C, and chelerythrine reduced cell proliferation to 7%, 31%, and 48% of control, respectively. Myristoylated peptide inhibition of PKCalpha and -epsilon reduced cell proliferation to 57% and 59% of control, respectively. PKCalpha antisense oligonucleotide reduced cell proliferation to 35% of control. Cyclin E protein level was decreased to 70%, 38%, 57%, and 43% of control in cells treated with calphostin C, staurosporine, chelerythrine, and PKCalpha antisense, respectively. CONCLUSIONS: PKC activity, in particular PKCalpha and -epsilon activity, is important in promoting corneal endothelial cell proliferation. Inhibition of PKC activity prohibits G1/S-phase progression and reduces cyclin E protein levels.     

Latent proliferative sickle cell retinopathy in sickle cell trait

PURPOSE: To describe a patient with sickle cell trait who developed latent proliferative sickle cell retinopathy after mild blunt trauma. METHOD: Case Report. A 20-year-old man with unilateral Stage 3 sickle retinopathy associated with an ischaemic ridge presenting three years after the initial mild blunt ocular trauma. RESULTS: Fundus examination of the left eye showed an ischaemic ridge delineating avascular from vascular retina. Fluorescein angiography of the left eye showed an avascular peripheral retina and multiple sea fan neovascularization. Blood studies showed him to be Hb AS. CONCLUSIONS: In our patient the proliferative changes were the result of his initial mild trauma associated with an increase in the intraocular pressure. The latent development of the sea-fan neovascularization associated with an ischaemic ridge is unusual. Advice about potential complications to patients with Hb AS after ocular trauma is advocated.     

玻璃体内细胞移植或细胞衍生物注入在视神经损伤中的作用

视网膜神经节细胞绝大部分位于神经节细胞层,可通过视神经通路将光感受器接收的视觉信息传递至高级视觉中枢,产生视觉。视网膜神经节细胞损伤和视神经轴突的中断,往往会导致视力下降甚至失明。近年来研究证实,玻璃体内细胞移植对损伤的视神经具有保护作用,为视神经损伤的修复治疗提供了新的方向。本文就玻璃体内细胞移植或细胞衍生物注入在视神经损伤中的作用展开综述。     

Squamous cell carcinoma of cornea

Purpose To report a case of isolated squamous cell carcinoma of cornea. Methods It is an observational case report. Results An 80-year-old man presented with decreased vision and a white gelatinous mass on his left cornea. Ocular examination revealed sparing of the limbus. There was no lymphadenopathy and no evidence of metastasis. The mass was excised completely with superficial keratectomy. Histopathology of the mass revealed it to be an invasive well-differentiated squamous cell carcinoma of cornea. Postoperatively mitomycin C (0.002%) eye drops were prescribed for 4 weeks. There has been no recurrence of the lesion till date. Conclusions Isolated squamous cell carcinoma of cornea, though a rare entity, should be kept in mind in patients with any corneal mass.