多聚二磷酸腺苷核糖聚合酶-1通过激活NF-κB途径导致高糖人视网膜血管内皮细胞凋亡

目的　探讨多聚二磷酸腺苷核糖聚合酶-１［ｐｏｌｙ（ａｄｅｎｏｓｉｎｅｄｉｐｈｏｓｐｈａｔｅ-ｒｉｂｏｓｅ）ｐｏｌｙｍｅｒａｓ-１,ＰＡＲＰ-１］和核因子-κＢ（ｎｕ-ｃｌｅａｒｆａｃｔｏｒ-κＢ,ＮＦ-κＢ）在高糖人视网膜血管内皮细胞（ｒｅｔｉｎａｌｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｃｅｌｌｓ,ＲＶＥＣ）中的相互作用及其在人ＲＶＥＣ中的表达定位及作用机制。方法　体外培养人ＲＶＥＣ及ＨＥＫ２９３Ｔ细胞,并进行传代,同时构建高糖人ＲＶＥＣ细胞模型。构建ＰＡＲＰ-ＥＧＦＰ及Ｆｌａｇ-ＮＦ-κＢ质粒,酶切鉴定后转染高糖培养的人ＲＶＥＣ,Ｗｅｓｔｅｒｎｂｌｏｔ法检测重组质粒表达目的基因的效果,并应用Ｗｅｓｔｅｒｎｂｌｏｔ法和免疫共沉淀法检测高糖人ＲＶＥＣ中ＰＡＲＰ-１和ＮＦ-κＢ的相互作用。ＰＡＲＰ-ＥＧＦＰ和Ｆｌａｇ-ＮＦ-κＢ质粒共转染人ＲＶＥＣ,激光共聚焦扫描显微镜检测ＰＡＲＰ-１和ＮＦ-κＢ在高糖人ＲＶＥＣ中的定位及其相互作用。结果　人ＲＶＥＣ复苏后２４ｈ贴壁,细胞呈扁平梭形,３ｄ左右细胞开始融合呈铺路石样,单层生长,铺满瓶底,并可见接触抑制现象。成功构建ＰＡＲＰ-ＥＧ-ＦＰ及Ｆｌａｇ-ＮＦ-κＢ质粒,并有效表达目的基因。免疫共沉淀结果显示ＰＡＲＰ-１和ＮＦ-κＢ是相互作用的蛋白质,高糖组ＮＦ-κＢｐ５０的条带比正常对照组明显增粗,并且高糖情况下ＰＡＲＰ-１结合ＮＦ-κＢ的量较正常对照组明显增加。激光共聚焦扫描显微镜结果显示ＰＡＲＰ和ＮＦ-κＢ均表达于正常的人ＲＶＥＣ的细胞核和核周区域,当受到高浓度葡萄糖影响后,ＰＡＲＰ-１和ＮＦ-κＢ均集中表达于细胞核内,尤以ＮＦ-κＢ最显著。结论　ＰＡＲＰ-１和ＮＦ-κＢ是相互作用的蛋白质,血糖增高时,ＰＡＲＰ-１可能进入细胞核内并结合同时进入细胞核的ＮＦ-κＢ,激活ＮＦ-κＢ信号通路,引起ＲＶＥＣ凋亡,导致ＤＲ的发生。     

SN50对大鼠视网膜缺血再灌注损伤的保护作用

目的　探讨核因子-κＢ（ｎｕｃｌｅａｒｆａｃｔｏｒ-κＢ,ＮＦ-κＢ）活性抑制剂ＳＮ５０对大鼠视网膜缺血再灌注损伤（ｒｅｔｉｎａｌｉｓｃｈｅｍｉｃａｌｒｅｐｅｒｆｕｓｉｏｎｉｎｊｕｒｙ,ＲＩＲＩ）的保护作用。方法　２７只成年雄性ＳＤ大鼠随机分为正常对照组、ＲＩＲＩ组、ＲＩＲＩ＋ＳＮ５０组,每组９只,建立ＲＩＲＩ动物模型,ＲＩＲＩ后６ｈ、２４ｈ、７２ｈＨＥ染色观察大鼠视网膜病理变化,免疫组织化学检测ＮＦ-κＢ的表达,实时定量ＰＣＲ检测ＴＮＦ-α的表达。结果　ＲＩＲＩ组ＲＩＲＩ６ｈ后视网膜出现组织轻度水肿,少量细胞变性,随着时间的延长,视网膜的损伤逐渐加重;ＲＩＲＩ＋ＳＮ５０组视网膜的损伤明显减轻。ＲＩＲＩ后６ｈ视网膜上可见ＮＦ-κＢ阳性表达,经过ＳＮ５０注射处理后,ＮＦ-κＢ的阳性表达在ＲＩＲＩ后２４ｈ明显减弱;经过ＳＮ５０注射处理后,与ＲＩＲＩ组比较,２４ｈ后视网膜的ＴＮＦ-α表达差异有统计学意义（Ｐ＜０．０５）。结论　ＳＮ５０对ＲＩＲＩ具有保护作用。     

视网膜下液生长因子含量与增生性玻璃体视网膜病变关系

目的 探讨生长因子与增生性玻璃体视网膜病变形成和发展的关系。方法 选４１例孔源性视网膜脱离伴ＰＶＲ患者,采用放射免疫方法测其ＴＮＦ,ＦＧＦ,ＴＧＦ－β,ＥＧＦ的含量。结果 ＰＶＲ患者视网膜下液中ＴＮＦ,ＥＧＦ,ＦＧＦ浓度明显高于对照组（Ｐ〈０．０５）。ＴＮＦ,ＥＧＦ,ＦＧＦ浓度Ｃ组高于Ｂ组,Ｂ组高于Ａ组,差异有显著性（Ｐ〈０．０５）。视网膜下液中ＴＮＦ,ＥＧＦ浓度各组高于血浆中ＴＮＦ,ＥＧＦ浓度,     

通心络胶囊对糖尿病小鼠视网膜IKKβ/IKBα/NF-κB通路的作用

目的　观察通心络胶囊对糖尿病小鼠视网膜ＩＫＫβ／ＩＫＢα／ＮＦ-κＢ通路的影响。方法　４０只ＫＫ／Ｕｐｊ-Ａｙ小鼠随机分为模型组、通心络低（１ｇ?ｋｇ－１）、中（２ｇ?ｋｇ－１）、高（４ｇ?ｋｇ－１）剂量组,每组各１０只,１０只Ｃ５７ＢＬ／６小鼠为对照组。通心络组按相应剂量灌胃给药,对照组和模型组给予等体积的蒸馏水,每天１次,连续１２周。测定体质量、空腹血糖值（ｆａｓｔｉｎｇｂｌｏｏｄｇｌｕ-ｃｏｓｅ,ＦＢＧ）,伊文思蓝法（ｅｖａｎｓｂｌｕｅｍｅｔｈｏｄ,ＥＢ）测定血-视网膜屏障的通透性,ＨＥ染色法观察视网膜形态学变化,Ｒｅａｌ-ｔｉｍｅＰＣＲ（ｑＰＣＲ）和Ｗｅｓｔｅｒｎｂｌｏｔ法测定ＩＫＫβ、ＩＫＢα、ＮＦ-κＢｍＲＮＡ和蛋白的表达及Ｐ-ＩＫＢα和核ＮＦ-κＢ蛋白的表达。结果　通心络高剂量组较中、低剂量组细胞结构更致密,排列更有序。通心络低、中、高剂量组ＥＢ含量分别（２０．８２±３．８８）ｎｇ?ｍＬ－１、（１６．４３±２６０）ｎｇ?ｍＬ－１、（１６．１４±２．４２）ｎｇ?ｍＬ－１,较模型组（２５．５３±３．５７）ｎｇ?ｍＬ－１明显下降（均为Ｐ＜０．０５）。通心络中、高剂量组ＩＫＫβｍＲＮＡ和蛋白表达量较模型组显著下降（均为Ｐ＜０．０１）;通心络组ＩＫＢαｍＲＮＡ,通心络中、高剂量组ＩＫＢα蛋白以及Ｐ-ＩＫＢα蛋白的表达均较模型组显著下降（均为Ｐ＜０．０１）,通心络低、中、高剂量组核ＮＦ-κＢ蛋白表达量为０．５２±０．０５、０４３±００５、０３４±０．０４,显著低于模型组（０．６１±０．０７）（均为Ｐ＜０．０５）。结论　通心络胶囊可明显改善ＫＫ／Ｕｐｊ-Ａｙ小鼠视网膜病变,其机制与抑制ＩＫＫβ／ＩＫＢα／ＮＦ-κＢ通路相关。     

脉络膜新生血管模型大鼠视网膜核转录因子-κBp65、血管内皮生长因子和碱性成纤维细胞生长因子表达的变化

目的　观察脉络膜新生血管（ｃｈｏｒｏｉｄａｌｎｅｏｖａｓｃｕｌａｒｉｚａｔｉｏｎ,ＣＮＶ）模型大鼠视网膜核转录因子-κＢｐ６５（ｎｕｃｌｅａｒｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒｋａｐｐａ-Ｂｐ６５,ＮＦ-κＢｐ６５）、血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）、碱性成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ,ｂＦＧＦ）表达的变化。方法　取ＢＮ大鼠１２只,随机分为正常组、模型组,每组各６只。正常组不做任何处理,模型组以１００ｇ·Ｌ-１水合氯醛腹腔注射麻醉,复方托吡卡胺滴眼液双眼散瞳,倍诺喜行眼表麻醉,在全视网膜镜下用５３２ｎｍ激光于视盘周围做视网膜光凝建立ＣＮＶ模型。每周行ＦＦＡ、ＯＣＴ检查眼底,观察ＣＮＶ生成情况。５周后处死大鼠,取大鼠视网膜用免疫组织化学法检测视网膜内ＮＦ-κＢｐ６５、ＶＥＧＦ、ｂＦＧＦ阳性表达积分光密度。结果　造模５周后ＦＦＡ及ＯＣＴ检查可见模型组ＣＮＶ形成。免疫组织化学法检测结果:正常组大鼠视网膜上ＮＦ-κＢｐ６５阳性表达的积分光密度为９２６４．３３±１４７９．４９,模型组的积分光密度为３４８１５．８３±３８７３．６１,两组比较差异有统计学意义（ｔ＝１５．０９５,Ｐ＜０．０１）;正常组大鼠视网膜上ＶＥＧＦ阳性表达的积分光密度为１９９４．９３±３０９．００,模型组为１３３１８．５４±１９５８．１１,两组比较差异有统计学意义（ｔ＝１３．９９２,Ｐ＜０．０１）;正常组大鼠视网膜上ｂＦＧＦ阳性表达的积分光密度为１６０８．７４±２３５．５５,模型组为１５９６３．１４±２０３４．１２,两组比较差异有统计学意义（ｔ＝１７．１７１,Ｐ＜０．０１）。结论　５３２ｎｍ激光光凝可诱导大鼠视网膜上ＮＦ-κＢｐ６５的表达,ＮＦ-κＢｐ６５在ＣＮＶ发生发展中可能起重要作用。     

莱菔硫烷对链脲佐霉素诱导的大鼠糖尿病白内障的治疗作用

目的　探讨莱菔硫烷（Ｓｕｌｆｏｒａｐｈａｎｅ,ＳＦＮ）对链脲佐霉素诱导的大鼠糖尿病白内障的治疗作用。方法　将７２只雄性ＳＤ大鼠随机分为正常对照组、模型组、ＳＦＮ低剂量干预组、ＳＦＮ中剂量干预组、ＳＦＮ高剂量干预组和苄达赖氨酸（ｂｅｎｄａｚａｃｌｙ-ｓｉｎｅ,ＢＤＬ）组,每组１２只,后５组腹腔一次性注射链脲佐菌素（６５ｍｇ·ｋｇ－１）建立糖尿病白内障模型,从造模成功当日开始,ＳＦＮ低、中、高剂量干预组分别给予５０ｍｇ·ｋｇ－１、１００ｍｇ·ｋｇ－１、２００ｍｇ·ｋｇ－１ＳＦＮ灌胃,ＢＤＬ组以２００ｍｇ·ｋｇ－１ＢＤＬ灌胃,其余２组用同体积的生理盐水,每天１次,治疗１２周后,测定空腹血糖水平,裂隙灯观察大鼠晶状体变化并对其混浊度进行分级,分离晶状体并制备成匀浆液,测定丙二醛（ｍａｌｏｎｄｉａｌｄｅｈｙｄｅ,ＭＤＡ）含量及超氧化物歧化酶（ｓｕｐｅｒｏｘｉｄｅｄｉｓｍｕｔａｓｅ,ＳＯＤ）、谷胱甘肽过氧化物酶（ｇｌｕｔａｔｈｉｏｎｅ-ｐｅｒｏｘｉｄａｓｅ,ＧＳＨ-Ｐｘ）、过氧化氢酶（ｃａｔａｌａｓｅ,ＣＡＴ）活性,ＥＬＩＳＡ检测糖基化终末产物（ａｄｖａｎｃｅｄｇｌｙｃｏｓｙｌａｔｉｏｎｅｎｄｐｒｏｄｕｃｔｓ,ＡＧＥｓ）含量,行Ｗｅｓｔｅｒｎ-ｂｌｏｔｔｉｎｇ检测醛糖还原酶（ａｌｄｏｓｅｒｅｄｕｃｔａｓｅ,ＡＲ）表达。结果　用药后１２周,ＳＦＮ中、高剂量干预组大鼠空腹血糖明显低于模型组（均为Ｐ＜０．０５）,而ＳＦＮ低剂量干预组、ＢＤＬ组仍处于高血糖水平,与模型组比较,差异均无统计学意义（均为Ｐ＞０．０５）。正常对照组大鼠晶状体透明,无混浊发生;而模型组大鼠晶状体出现雾状混浊、核混浊,分级为Ⅲ ～Ⅴ级,其混浊度显著高于正常对照组（Ｐ＜０．０１）。与模型组比较,ＳＦＮ中、高剂量干预组晶状体混浊度等级差异明显减轻（均为Ｐ＜０．０５）,而ＳＦＮ低剂量干预组、ＢＤＬ组晶状体混浊度等级与模型组相比,差异均无统计学意义（均为Ｐ＞０．０５）。与正常对照组比较,模型组大鼠晶状体组织中ＭＤＡ含量升高,而ＳＯＤ、ＧＳＨ-Ｐｘ、ＣＡＴ活性降低,差异均有统计学意义（均为Ｐ＜０．０１）。经中、高剂量ＳＦＮ或ＢＤＬ处理后,ＭＤＡ含量较模型组减少（均为Ｐ＜０．０５）,ＳＯＤ、ＧＳＨ-Ｐｘ、ＣＡＴ活性较模型组增加（均为Ｐ＜０．０５）。而ＳＦＮ低剂量干预组上述指标与模型组相比,差异均无统计学意义（均为Ｐ＞０．０５）。模型组大鼠晶状体组织中ＡＧＥｓ含量较正常对照组升高（Ｐ＜０．０１）;相对于模型组,经中、高剂量ＳＦＮ处理１２周后,ＡＧＥｓ含量明显减少（均为Ｐ＜０．０５）;而ＳＦＮ低剂量干预组、ＢＤＬ组ＡＧＥｓ含量与模型组比较,差异均无统计学意义（均为Ｐ＞０．０５）。运用Ｗｅｓｔｅｒｎ-ｂｌｏｔｔｉｎｇ检测各组大鼠晶状体组织ＡＲ蛋白表达,结果显示:正常对照组ＡＲ蛋白呈低水平表达,模型组ＡＲ蛋白表达明显上调,与正常对照组比较,差异有统计学意义（Ｐ＜０．０１）;相对于模型组,经中、高剂量ＳＦＮ或ＢＤＬ处理后,ＡＲ蛋白表达水平降低（均为Ｐ＜０．０５）,而ＳＦＮ低剂量干预组ＡＲ蛋白表达水平与模型组比较,差异无统计学意义（Ｐ＞０．０５）。结论　ＳＦＮ对链脲佐菌素诱导的大鼠糖尿病白内障有治疗作用,其机制可能与增强抗氧化能力、抑制ＡＧＥｓ产生及下调ＡＲ表达有关。     

核转录因子-κB在氧诱导血管增殖性视网膜病变小鼠中的表达

目的　探讨氧诱导血管增殖性视网膜病变小鼠视网膜中核转录因子（ｎｕｃｌｅａｒｆａｃｔｏｒ,ＮＦ）-κＢ蛋白的表达变化及意义。方法　７２只出生后７ｄ的Ｃ５７ＢＬ／６Ｊ小鼠随机分为实验组（３６只）和对照组（３６只）,实验组与母鼠一起放入氧气含量为体积分数７５％的饲养箱中饲养,出生后１２ｄ回到正常大气环境中;对照组小鼠始终在正常大气环境中饲养。分别于出生后１２ｄ、１４ｄ、１７ｄ时处死小鼠,荧光素灌注后行视网膜铺片观察血管分布及形态,Ｗｅｓｔｅｒｎｂｌｏｔ法检测视网膜中血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）的蛋白表达变化,免疫组织化学染色检测眼球中ＮＦ-κＢ的表达变化。结果　视网膜铺片检查结果可见,实验组小鼠出生后１４ｄ开始出现视网膜新生血管,面积为每眼（０. ０６±０．１２）ｍｍ２;生后１７ｄ达到高峰,面积为每眼（１．７９±０．０２）ｍｍ２（χ２＝２０．１１２,Ｐ＜０．０１）。Ｗｅｓｔｅｒｎｂｌｏｔ检测结果显示,实验组ＶＥＧＦ表达不断增强,在生后１７ｄ时表达达到高峰,为０．８３±０．０５。免疫组织化学染色结果显示,实验组生后１２ｄＮＦ-κＢ阳性细胞计数为（１２．３２±１. ０４）个?ｍｍ－２,１４ｄ时为（１９．１６±１．０２）个?ｍｍ－２,生后１７ｄ阳性表达较１２ｄ和１４ｄ时均高,为（２８. ６０±０．５２）个?ｍｍ－２（χ２ ＝１３. １０２,Ｐ＜０．０５;χ２＝２０．１３２,Ｐ＜０．０１）。结论　ＮＦ-κＢ在氧诱导血管增殖性视网膜病变小鼠视网膜中表达明显升高,其趋势与ＶＥＧＦ的变化相同,ＮＦ-κＢ参与了缺氧状态下视网膜新生血管形成的调控过程。     

视网膜静脉阻塞患者血浆和泪液中血管内皮生长因子表达的研究

目的　观察视网膜静脉阻塞（ｒｅｔｉｎａｌｖｅｉｎｏｃｃｌｕｓｉｏｎ,ＲＶＯ）患者血浆和泪液中血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）的表达情况。方法　选取我院确诊的２８例ＲＶＯ患者,收集血浆和泪液样本,通过酶联免疫吸附试验测定ＶＥＧＦ表达水平,在１ｄ、２周和４周使用光学相干断层扫描检查中央视网膜厚度（ｃｅｎｔｒａｌｒｅｔｉｎａｌｔｈｉｃｋｎｅｓｓ,ＣＲＴ）,另选健康志愿者３０人作为对照组,比较两组血浆和泪液中ＶＥＧＦ、ＣＲＴ表达差异。结果　各时间点,ＲＶＯ组血浆中ＶＥＧＦ表达水平显著高于对照组（均为Ｐ＜０．０５）,１ｄＲＶＯ组泪液ＶＥＧＦ表达水平与对照组差异无统计学意义（Ｐ＞０．０５）。ＲＶＯ组血浆和泪液中ＶＥＧＦ表达水平呈微弱正相关（Ｐ＜０．０５）,而对照组中这种相关性稍强（Ｐ＜０．０５）。两组２周后ＣＲＴ明显增加,１ｄ与２周ＣＲＴ值差异均有统计学意义（均为Ｐ＜０．０５）,２周与４周差异均无统计学意义（均为Ｐ＞０．０５）,各时间段ＲＶＯ组与对照组差异有统计学意义（Ｐ＜０．０５）。结论　ＲＶＯ患者泪液中ＶＥＧＦ表达水平及ＣＲＴ与健康人比较显著升高,血浆与泪液中ＶＥＧＦ表达水平的变化有一致性,泪液检测作为非侵入性的检测方式,对ＲＶＯ的早期诊断有较高的临床应用价值。     

玻璃体内注射色素上皮源性因子对大鼠早期糖尿病视网膜病变的保护作用

目的　观察玻璃体内注射色素上皮源性因子（ｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ-ｄｅｒｉｖｅｄｆａｃｔｏｒ,ＰＥＤＦ）对ＳＤ糖尿病大鼠视网膜ＰＥＤＦ、血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）、炎性相关因子细胞间黏附分子-１（ｉｎｔｅｒｃｅｌｌｕｌａｒｃｅｌｌａｄｈｅｓｉｏｎｍｏｌｅ-ｃｕｌｅ-１,ＩＣＡＭ-１）和单核细胞趋化蛋白-１（ｍｏｎｏｃｙｔｅｃｈｅｍｏｔａｃｔｉｃｐｒｏｔｅｉｎ-１,ＭＣＰ-１）以及细胞通透性相关因子紧密连接蛋白-１（ｚｏｎｕ-ｌａｏｃｃｌｕｄｅｎｓ-１,ＺＯ-１）和蛋白激酶Ｂ（ｐｒｏｔｅｉｎｋｉｎａｓｅＢ,ＰＫＢ,又称Ａｋｔ）表达的影响,探讨ＰＥＤＦ对早期糖尿病视网膜病变的保护作用。方法　选取６～８周龄ＳＤ大鼠６０只,随机分为４组:正常对照组（ＣＯＮ组）、糖尿病组（ＤＭ组）、糖尿病生理盐水注射组（ＤＭ＋ＮＳ组）和糖尿病ＰＥＤＦ注射组（ＤＭ＋ＰＥＤＦ组）。链脲佐菌素诱导建立糖尿病模型后第１周、２周、３周时,ＤＭ＋ＰＥＤＦ组大鼠玻璃体内注射ＰＥＤＦ,而ＤＭ＋ＮＳ组注射同样体积的生理盐水。第４周时处死大鼠,摘出眼球,进行视网膜组织病理学及电镜观察。并采用免疫组织化学方法检测视网膜ＰＥＤＦ、ＶＥＧＦ、ＩＣＡＭ-１、ＭＣＰ-１以及ＺＯ-１、Ａｋｔ的表达情况。结果　糖尿病大鼠均造模成功。４周糖尿病病程尚不能导致明显的视网膜新生血管形成。在透射电镜下,ＤＭ组大鼠视网膜可见神经节细胞水肿,线粒体肿胀变大,基质肿胀明显,可见大部分或全部的嵴消失,部分双侧膜融合,粗面内质网扩张,而玻璃体内ＰＥＤＦ注射可以明显地改善这一病理改变。ＤＭ组大鼠ＰＥＤＦ主要表达于视网膜神经纤维层、神经节细胞层以及内丛状层、光感受器基质以及色素上皮层、脉络膜等部位;ＶＥＧＦ主要表达于神经节细胞层,ＩＣＡＭ-１主要表达在光感受器层,ＭＣＰ-１主要表达在视网膜内层细胞,包括节细胞层和内核层,ＺＯ-１蛋白主要表达于内核层的细胞以及神经节细胞,Ａｋｔ主要表达于内丛状层以及脉络膜的细胞浆中。同ＣＯＮ组相比,ＤＭ组、ＤＭ＋ＮＳ组视网膜中ＰＥＤＦ、ＶＥＧＦ表达差异均无统计学意义（均为Ｐ＞０．０５）,而ＩＣＡＭ-１、ＭＣＰ-１表达增加,ＺＯ-１、Ａｋｔ表达减少,差异均有统计学意义（均为Ｐ＜０．０５）。ＤＭ＋ＰＥＤＦ组大鼠视网膜中ＰＥＤＦ、ＶＥＧＦ的表达较ＤＭ组和ＤＭ＋ＮＳ组差异均无统计学意义（均为Ｐ＞０．０５）,但ＩＣＡＭ-１、ＭＣＰ-１表达减少,ＺＯ-１、Ａｋｔ表达增多,差异均有统计学意义（均为Ｐ＜０．０５）。结论　ＰＥＤＦ可以明显地改善糖尿病视网膜病变早期视网膜神经节细胞的损害,通过减少炎性因子和增加细胞连接蛋白而减轻血-视网膜屏障的破坏,从而对早期糖尿病视网膜病变起一定的防治作用。     

TGF-β介导的Smad信号通路在增生型糖尿病视网膜病变中的作用和意义

目的　本研究旨在观察转化生长因子β（ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒ-ｂｅｔａ,ＴＧＦ-β）介导的Ｓｍａｄ信号通路在增生型糖尿病视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅｄｉａｂｅｔｉｃｒｅｔｉｎｏｐａｔｈｙ,ＰＤＲ）中的作用和意义,进而阐明其可能的发病机制。方法　收集正常人和糖尿病患者的血液标本,将糖尿病患者依据视网膜受累程度分为２组:无糖尿病视网膜病变（ｎｏｎ-ｄｉａｂｅｔｉｃｒｅｔｉ-ｎｏｐａｔｈｙ,ＮＤＲ）组、糖尿病视网膜病变（ｄｉａｂｅｔｉｃｒｅｔｉｎｏｐａｔｈｙ,ＤＲ）组,根据眼底改变又分为非增生型糖尿病视网膜病变（ｎｏｎ-ｐｒｏｌｉｆｅｒａｔｉｖｅｄｉａｂｅｔｉｃｒｅｔｉｎｏｐａｔｈｙ,ＮＰＤＲ）组和增生型糖尿病视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅｄｉａｂｅｔｉｃｒｅｔｉｎｏｐａｔｈｙ,ＰＤＲ）组。分别用ＥＬＩＳＡ检测正常人和糖尿病患者血浆中的ＴＧＦ-β１ 和ＴＧＦ-β２,Ｗｅｓｔｅｒｎｂｌｏｔ检测血浆中ＴＧＦ-β１、ＴＧＦ-β２、Ｓｍａｄ３和ｐ-Ｓｍａｄ３的蛋白表达量,比较各组差异。结果　与对照组比较,糖尿病患者各组ＴＧＦ-β１ 和ＴＧＦ-β２分泌量和ＴＧＦ-β１、ＴＧＦ-β２、ｐ-Ｓｍａｄ３的蛋白表达量均明显升高（均为Ｐ＜０．０５）;与ＮＤＲ组比较,ＤＲ各组上述指标均显著增高（均为Ｐ＜０．０５）;ＰＤＲ组上述指标均显著高于ＮＰＤＲ组（均为Ｐ＜０．０５）。结论　ＴＧＦ-β介导的Ｓｍａｄ信号通路参与了ＤＲ的发生,可能是介导ＰＤＲ发生的重要机制。     

Neuroprotection and regeneration after traumatic lesion of the optic nerve

BACKGROUND: After a traumatic lesion of the optic nerve, retinal ganglion cells (RGC) undergo massive degeneration by apoptosis, which leads to loss of vision in the affected eye. Like other neurones in the central nervous system, RGC are not able to regenerate their damaged axons spontaneously. We used special surgical methods and pharmacological measures to achieve enhanced survival and regeneration of damaged RGC. MATERIALS AND METHODS: Studies were performed using the model of RGC degeneration induced by severing the optic nerve of adult rats. RGC were loaded with a fluorescent dye, and several drugs were applied intravitreally. The effects were evaluated after two weeks by counting the surviving RGC. For regeneration studies, an autologous peripheral nerve graft was sutured to the stump of the cut optic nerve, or the ends of the cut optic nerve were re-sutured. Recovery of RGC function was assessed by VEP measurements. RESULTS: The number of RGC surviving an axotomy increased significantly after intravitreal injections of aurintricarboxylic acid, cortisol, a caspase inhibitor, brimonidine or microglia-targeted substances. Regeneration of cut axons was enhanced by aurintricarboxylic acid or cortisol. In addition, considerable neuroprotective and regenerative effects including partial restoration of VEP were induced by lens injury, which results in a gradual release of crystallins into the vitreous, or by intravitreal injection of purified crystallins. CONCLUSION: The loss of vision after an optic nerve trauma can be reduced in this animal model by suitable neuroprotective measures, which raises hope for the treatment of patients.     

大鼠视神经挫伤视网膜形态功能变化的动态研究

目的观察视神经夹挫伤后视网膜形态学和视功能动态变化,为视功能评价和视神经保护研究提供依据。方法大鼠视神经夹挫伤后1d、3d、5d7、d、9d、2周4、周8、周1、2周,光镜观察视网膜神经节细胞(RGC)改变,闪光视觉诱发电位(F-VEP)检测视功能状况。结果视神经部分损伤后3d到1周内视网膜神经节细胞快速减少,2周以后缓慢减少,4周几乎无明显变化;视神经损伤1d,F-VEP波形变得低而宽,前2周呈进行性下降期,4周后变化平稳,并显示恢复迹象。结论神经节细胞继发性损伤是视功能进行性下降的重要原因,一定数量存活的视网膜节细胞是视功能恢复的基础;神经损伤变化和视功能变化与时间具有一定的相关性,这些对于正确评价视功能状况和预后有极重要的意义。     

Neuroprotective effect of latanoprost on rat retinal ganglion cells

Background To investigate the neuroprotective effect of intravitreal administration of latanoprost on retinal ganglion cell (RGC) damage induced by N-methyl-D-aspartic acid (NMDA) or optic nerve axotomy.Methods Using Sprague-Dawley rats, retinal ganglion cell damage was induced by either intravitreal administration of NMDA or optic nerve axotomy. Latanoprost at doses of 0.03, 0.3, 3, 30 and 300 pmol was administered intravitreally before NMDA injection or optic nerve axotomy. Retinal damage was evaluated by counting the number of surviving RGCs retrogradely labeled with fluorogold under the microscope.Results Seven days after the NMDA injury, the number of surviving RGCs was significantly increased at doses of more than 30 pmol atanoprost (846±178 cells/mm² P=0.0166) compared with vehicle control (556±122 cells/mm²). Ten days after the optic nerve axotomy, the number of surviving RGC was significantly increased even at a dose of 0.3 pmol (815±239 cells/mm², P=0.0359) compared with control (462±75 cells/mm²).Conclusions Intravitreal administration of latanoprost has a neuroprotective effect on rat RGC damage induced by either NMDA or optic nerve axotomy, while its pharmacological features are different.     

促红细胞生成素缓释微球玻璃体腔注射对视网膜神经节细胞的保护作用

目的 探讨乳酸/羟基乙酸共聚物（PLGA）装载的促红细胞生成素（EPO）缓释微球（EPO-PLGA微球）经玻璃体腔注射对大鼠视神经挫伤模型中受损视网膜神经节细胞（RGC）的保护作用.方法 选取成年SD大鼠,建立视神经挫伤模型.建模后分别经玻璃体腔内注射含10 IU EPO的PLGA微球（EPO-PLGA组）、10 IU EPO（EPO组）、5 μl空白PLGA（PLGA组）、5 μl PBS（PBS组）,另设未治疗组不予玻璃体腔注药.术后5 d和2周,做视网膜切片,对各组RGC凋亡情况行TUNEL检测;术后23 d,DiI上丘逆标RGC,并于术后4周处死大鼠,视网膜铺片观察各组RGC存活情况;每组各个时间点分别处死6只SD大鼠.采用方差分析对结果进行比较.结果 TUNEL检测显示,术后5 d和2周,各组均可见TUNEL阳性细胞,其中EPO-PLGA组和EPO组TUNEL阳性细胞显著减少,其细胞凋亡率明显少于PLGA组、PBS组及未治疗组.术后4周,视网膜铺片RGC计数显示,正常SD大鼠RGC密度为（2387.7±164.9）个/mm^2,未治疗组为（748.3±58.8）个/mm^2,EPO-PLGA组为（1296.7±157.6）个/mm^2,EPO组为（1418.5±154.9）个/mm^2,PLGA组为（821.7±52.1）个/mm^2,PBS组为（804.4±86.4）个/mm^2;可见EPO-PLGA组和EPO组较未治疗组细胞密度显著增高,具有明显的RGC保护作用（P均＜0.01）,而EPO-PLGA组和EPO组间差异无统计学意义（P=0.065）.结论 EPO-PLGA缓释微球与EPO具有等效的RGC保护作用,这为进一步观察EPO-PLGA缓释微球的长效神经保护作用奠定了基础.     

超声微泡造影剂联合美金胺增强视神经损伤大鼠视网膜神经节细胞保护作用

目的 探讨超声微泡造影剂联合美金胺对视神经损伤大鼠视网膜神经节细胞( RGC)的保护作用.方法 将Sprague-Dawley(SD)雄性成年大鼠40只随机分为正常对照组(A组),假手术组(B组),空白对照组(C组),玻璃体腔单独注射美金胺组(D组),玻璃体腔注射美金胺加超声微泡组(E组)5个组,每组8只大鼠,再将各组随机分为视神经损伤后1、2周2个亚组,各亚组4只大鼠.A组不做任何处理;B组只暴露视神经,不进行钳夹,玻璃体腔注射生理盐水,立即用超声波辐照大鼠眼球;C～E组建立视神经钳夹伤模型后,处理方式分别为C组玻璃体腔注射生理盐水,D组玻璃体腔注射美金胺,E组玻璃体腔注射超声微泡造影剂及美金胺,立即用超声波辐照大鼠眼球.视神经损伤1、2周时,各组行逆行荧光金标记RGC并计数;闪光视觉诱发电位(F-VEP)检测,记录P100波潜伏期及振幅;荧光电子显微镜下观察视网膜细胞形态学改变.结果 逆行荧光金标记RGC结果显示,各处理组视网膜定向铺片上均可见金黄色着染的RGC.A、B组RGC数间差异无统计学意义(q=0.018,0.011;P=0.986,0.873);C～E组RGC数均较A组减少,差异具有统计学意义(F=85.944,P=0.012);D组RGC数多于C组,差异具有统计学意义(q=1.721,1.924;P=0.043,0.037);E组RGC数明显高于C、D组,差异具有统计学意义(q=1.128,1.482,P=0.027,0.008;q=1.453,1.855,P=0.031,0.010).F-VEP检测发现,A、B组P100波潜伏期及振幅间差异无统计学意义(q=0.008,0.019,P=0.981,0.946;q=0.072,0.052,P=0.737,0.851) ;C～E组P100波潜伏期较A组延长,振幅较A组降低,差异具有统计学意义(F=134.312,106.312;P=0.017,0.009).荧光电子显微镜下观察发现,A、B组大鼠视网膜各层结构完整,排列整齐,RGC排列紧密整齐,细胞核均匀深染,胞核大小一致.C～E组大鼠的视网膜不同程度水肿变厚,RGC有不同程度的排列紊乱,空泡化及细胞数目减少.结论 超声微泡造影剂联合美金胺能抑制视神经损伤后大鼠RGC的丢失,促进其视功能的恢复,对视神经损伤大鼠的RGC具有保护作用.     

脑源性神经营养因子对大鼠视网膜节细胞损伤的保护作用

目的：观察脑源性神经营养因子（brain-derived neurotrophic factor,BDNF)对Sprague-Dawly(SD)大鼠视神经损伤后视网膜节细胞（retinal ganglin cells,RGC)存活的作用。方法：将SD大鼠分为正常对照组,夹伤对照组,溶剂对照组,BDNF治疗组4组,每组20只眼。荧光金逆行标记RGC后7天,除正常组外行球后视神经夹伤,将100ng BDNF注入BDNF治疗组大鼠玻璃体腔内,分别于5,7,14,21,28天行RGC计数。结果：视神经夹伤后第7天RGC开始减少,14天时降至正常对照的70．2％,28天时降至40．5％。与正常组相比BDNF治疗组14天时RGC数开始减少,但7、14、21、28天RGC数均明显多于夹伤组（P＜0．01）。结论：在视神经夹伤后眼内注射BDNF能减少RGC的死亡,对RGC损伤有保护作用。     

The neuropeptide NAP provides neuroprotection against retinal ganglion cell damage after retinal ischemia and optic nerve crush

Background  NAP, an 8-amino acid peptide (NAPVSIPQ=Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) derived from activity-dependent neuroprotective protein (ADNP), plays an important role in neuronal differentiation and the survival of neurons in different pathological situations. We already discovered that NAP increases the survival of retinal ganglion cells (RGC) in vitro, and supports neurite outgrowth in retinal explants at femtomolar concentrations. The aim of this study was to investigate the effects of NAP on RGC survival after transient retinal ischemia and optic nerve crush. Methods  RGC of male Wistar rats were labelled retrogradely with 6 l FluoroGold injected stereotactically into both superior colliculi. Seven days later, retinal ischemia was induced by elevating the intraocular pressure to 120 mm Hg for 60 minutes or by crushing one optic nerve for 10 s after a partial orbitotomy. NAP was either injected intraperitoneally in the concentration of 100 mg/kg 1 day before, directly after, and on the first and the second days after damage, or intravitreally (0.05 or 0.5 μg/eye) directly after the optic nerve crush. Controls received the same concentrations of a control peptide. Densities of surviving RGC and activated microglial cells (AMC) were quantified in a masked fashion 10 days after damage by counting FluoroGold-labelled cells. Results  After retinal ischemia, intraperitoneal injections of NAP increased the number of surviving RGC by 40% (p < 0.005) compared to the control group. After optic nerve crush, NAP raised the number of surviving RGC by 31% (p = 0.07) when injected intraperitoneally and by 54% (p < 0.05) when administered intravitreally. Conclusions  NAP acts neuroprotectively in vivo after retinal ischemia and optic nerve crush, and may have potential in treating optic nerve diseases. Supported by the Ernst und Berta Grimmke Stiftung, Germany. IG is the incumbent of the Lily and Avraham Gildor Chair for the Investigation of Growth Factors and the Director of the Adams Super Center for Brain Research at Tel Aviv University and is the Chief Scientific Officer of Allon Therapeutics Inc., Vancouver, Canada. An erratum to this article can be found at     

A new calcium channel antagonist, lomerizine, alleviates secondary retinal ganglion cell death after optic nerve injury in the rat

PURPOSE: We investigated whether lomerizine, a new diphenylmethylpiperazine calcium channel blocker, exerted a neuroprotective effect on axonal or retinal damage induced by optic nerve injury in the rat. METHODS: A partial crush lesion was inflicted unilaterally on the optic nerve, 2 mm behind the globe, in adult Wistar albino rats. Animals were treated with the vehicle, 10 or 30 mg/kg lomerizine. Each solution was given orally twice daily for 4 weeks. One week before euthanization, Fluoro-Gold (FG) was injected into both superior colliculi to retrogradely label surviving retinal ganglion cells (RGCs). Approximately 1 month after the optic nerve injury, the retinal damage was assessed morphologically, and the optic nerve axons surrounding the initial lesion were examined histologically. RESULTS: The mean RGC density in the control group decreased to 65.9 +/- 1.32% of the contralateral eye, whereas the systemic application of 10 or 30 mg/kg of lomerizine significantly enhanced the RGC survival to 88.1 +/- 0.38% and 89.8 +/- 0.28%, respectively. Histological examination of damaged axons revealed no significant enhancement of the density or total number of axons of the retinal ganglion cells in the lomerizine-treated group. The crush force we employed caused no significant morphological differences in the retinal layers between the sham-operated animals and the animals from the experimental groups. CONCLUSIONS: Our findings suggest that lomerizine alleviates secondary degeneration of RGCs induced by an optic nerve crush injury in the rat, presumably by improving the impaired axoplasmic flow.     

超声微泡造影剂介导脑源性神经营养因子联合转染视网膜和视皮质对视神经损伤后视网膜神经节细胞的保护作用

　　目的　观察超声微泡造影剂介导脑源性神经营养因子（BDNF）联合转染大鼠视网膜和视皮质区细胞对视神经损伤后视网膜神经节细胞（RGC）的保护作用。方法　雄性Sprague-Dawley（SD）大鼠88只随机分为正常组（A组）、假手术组（B组）、空白对照组（C组）、单纯眼转染组（D组）、单纯脑转染组（E组）、联合转染组（F组）;A组8只大鼠,B～F组每组16只大鼠。建立钳夹视神经损伤模型,将B～F组大鼠随机分为视神经损伤1、2周亚组,各亚组8只大鼠。B、C组玻璃体腔和视皮质区分别注射磷酸盐缓冲液（PBS）,D、E组玻璃体腔和视皮质区分别注射BDNF质粒（pBDNF）微泡造影剂悬液,F组玻璃体腔和视皮质区同时注射pBDNF微泡造影剂悬液。D～F组注射pBDNF微泡微泡造影剂悬液后,立即用超声辐照相应转染部位。视神经损伤后1、2周,各组行逆行荧光金标记RGC计数;半胱氨酸蛋白酶-3(caspase-3）蛋白免疫组织化学染色,观察其阳性表达情况;图形视网膜电流图（PERG）检测,记录N95振幅。结果　荧光金标记RGC结果显示,各组均可见金黄色荧光散布于视网膜定向铺片上。A~F组间RGC计数差异有统计学意义（F=256.30,P＜0.01）;B~F组视神经损伤1、2周亚组间RGC计数差异也有统计学意义（F=6518,P＜0.01）。光学显微镜观察发现,A、B组大鼠视网膜均未见caspase-3蛋白阳性表达;C~F组均可见主要位于神经节细胞层的caspase-3蛋白阳性表达。PERG检测发现,A~F组间N₉₅振幅差异有统计学意义（F=121.56,P＜0.01）;B~F组视神经损伤1、2周亚组间N95振幅差异也有统计学意义（F=8238,P＜0.01）。结论　超声微泡造影剂介导BDNF联合转染视网膜和视皮质区细胞能抑制视神经损伤后RGC凋亡,提高RGC存活数,保护其视功能。      

Neuroprotective effect of sulfhydryl reduction in a rat optic nerve crush model

PURPOSE: The signaling of retinal ganglion cell (RGC) death after axotomy is partly dependent on the generation of reactive oxygen species. Shifting the RGC redox state toward reduction is protective in a dissociated mixed retinal culture model of axotomy. The hypothesis for the current study was that tris(2-carboxyethyl)phosphine (TCEP), a sulfhydryl reductant, would protect RGCs in a rat optic nerve crush model of axotomy. METHODS: RGCs of postnatal day 4 to 5 Long-Evans rats were retrogradely labeled with the fluorescent tracer DiI. At approximately 8 weeks of age, the left optic nerve of each rat was crushed with forceps and, immediately after, 4 muL of TCEP (or vehicle alone) was injected into the vitreous at the pars plana to a final concentration of 6 or 60 microM. The right eye served as the control. Eight or 14 days after the crush, the animals were killed, retinal wholemounts prepared, and DiI-labeled RGCs counted. Bandeiraea simplicifolia lectin (BSL-1) was used to identify microglia. RESULTS: The mean number of surviving RGCs at 8 days in eyes treated with 60 microM TCEP was significantly greater than in the vehicle group (1250 +/- 156 vs. 669 +/- 109 cells/mm(2); P = 0.0082). Similar results were recorded at 14 days. Labeling was not a result of microglia phagocytosing dying RGCs. No toxic effect on RGC survival was observed with TCEP injection alone. CONCLUSIONS: The sulfhydryl-reducing agent TCEP is neuroprotective of RGCs in an optic nerve crush model. Sulfhydryl oxidative modification may be a final common pathway for the signaling of RGC death by reactive oxygen species after axotomy.