丝裂原活化蛋白激酶通路在糖尿病视网膜病变中的作用

丝裂原活化蛋白激酶（MAPK）信号转导通路是糖尿病视网膜病变（DR）发生、发展的共同通路。新近研究发现在糖尿病患者中，MAPK通路可被高糖、血管活性物质和细胞因子等多种因素单独或协同激活，并在和其它信号转导途径交叉作用下，影响细胞因子的产生和改变各种视网膜细胞的增殖活性，最终导致DR。本文主要讨论MAPK通路在DR患者的作用机制，以期通过各种手段阻断MAPK通路来防治DR的发生、发展。     

大鼠视神经损伤后视网膜小胶质细胞表达的初步观察

目的 研究大鼠视神经损伤后视网膜中小胶质细胞的表达情况.方法 实验研究.选取30只成年雌性健康SD大鼠,按照随机数字表法分为实验组和对照组各15只,分别用于细胞计数、免疫组织化学及免疫印迹实验.实验组在眼球后约1.5 ～2.0 mm处行右眼视神经鞘内切断术,术后5d于视神经断端处用荧光金逆行标记视网膜节细胞,手术后7d处死取材.对照组小鼠右眼行视神经切断术并标记,2d后处死取材.视网膜做铺片用于计数.用免疫组织化学法于视网膜切片上行小胶质细胞的表面标记物Iba-1染色,观察小胶质细胞的形态及数量,同时应用免疫印迹法检测视网膜内Iba-1蛋白含量的变化.两组间比较采用非配对student t-检验进行统计学分析.结果 对照组视网膜中有少量小胶质(Iba-1阳性)细胞表达,并呈非活化状态.视神经切断7d后小胶质细胞明显增多且呈半活化状态,免疫印迹结果显示损伤后Iba-1蛋白表达量明显增加到对照组的2.3倍(t=7.669,P=0.001).视视神经切断7d后节细胞数量为(1182±64)个/mm2,明显减少至对照组的51％(t=23.850,P＜0.01).结论 大鼠视神经损伤后小胶质细胞表达增多且呈部分激活状态,可能是视网膜受损后自我保护的表现之一.     

p38丝裂原活化蛋白激酶信号通路在糖尿病性视网膜病变中的作用研究进展

丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）通路是细胞将信号从细胞膜传递到核的主要通路,它是细胞促增殖和传递应激信号的关键激酶.该家族包括细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）、c-Jun氨基端激酶（c-Jun N-terminal kinase,JNK）、p38MAPK等亚族,其中p38MAPK在糖尿病性视网膜病变（diabetic retinopathy,DR）的形成及发展中起着非常重要的作用.最近的研究显示,糖尿病状态下的高糖——蛋白激酶C（protein kinase C,PKC）通路、糖基化终产物、氧化应激、生长因子、渗透压、牵张刺激等都可激活MAPK家族,通过调节转化生长因子β、基质金属蛋白酶活性及葡萄糖转运体的表达等多种途径影响纤维连接蛋白、Ⅳ型胶原的生成,参与DR的发生,影响DR的进程.通过深入研究p38MAPK在DR中的作用,将有助于阐述DR的发病机制,阻断MAPK通路将可能成为治疗DR的新策略、新方法.     

p38丝裂原活化蛋白激酶信号转导通路与角膜新生血管

炎症是促进角膜新生血管生长的重要因素。p38丝裂原活化蛋白激酶信号转导通路通过调控IL-1β,IL-6和TNF-α等的表达,从而在炎症反应中发挥重要作用。p38抑制剂不仅抑制炎性介质的表达,而且阻断其功能,因此在抑制炎症反应中具有重大作用。从p38信号转导角度研究角膜新生血管的形成机制,是角膜新生血管机制研究的新方向。     

黄体酮在大鼠视神经损伤再生中的保护作用

目的　探讨黄体酮对外伤引起的视网膜及视神经损伤后再生的作用及机制。方法　８０只大鼠随机分为正常组（不作任何处理）、假损伤组（只暴露视神经,不夹伤）、损伤１组（行视神经不完全损伤处理,伤后即刻给予腹腔注射生理盐水）、损伤２组（行视神经不完全损伤处理,伤后１ｈ给予腹腔注射黄体酮注射液）。分别于损伤后１ｄ、３ｄ、７ｄ、１４ｄ将各组５只大鼠右眼球摘除,取视网膜组织,ＨＥ染色光学显微镜观察视网膜形态学变化,免疫组织化学染色观察视网膜组织中谷氨酸转运体-１（ｇｌｕｔａｍａｔｅｔｒａｎｓｐｏｒｔｅｒ-１,ＧＬＴ-１）及活化Ｐ３８-ＭＡＰＫ（ｐ-ｐ３８）的表达变化。结果　经黄体酮治疗的损伤２组各个时间点大鼠视网膜细胞核排列整齐,稀疏程度及空泡化较轻,视网膜形态改变轻微。免疫组织化学染色检测损伤１组视网膜ＧＬＴ-１在损伤后１ｄ、３ｄ、７ｄ、１４ｄ表达的平均光密度值分别为０．３６８±０．０３３、０．１５１±０．０２７、０．１０６±０．０３１和０．０７６±０．０２０,损伤２组分别为０．４６１±０．０１１、０．２３１±０．０２１、０．１３２±０．０２２和０．１０３±０．０１３,各时间点损伤２组较损伤１组均有所增加,差异均有统计学意义（均为Ｐ＜０．０５）。损伤１组视网膜ｐ-ｐ３８在损伤后１ｄ、３ｄ、７ｄ表达的平均光密度值分别为０．４５９±０．０２９、０．３２４±０．０２３和０．２０１±０．０２４,损伤２组分别为０．３３９±０．０１９、０．２４５±０．０１４和０．１５４±０．０３２,各时间点损伤２组较损伤１组均明显降低,差异均有统计学意义（均为Ｐ＜０．０５）。结论　黄体酮对外伤性视网膜及视神经损伤均具有保护作用。     

基于丝裂原活化蛋白激酶信号通路灯盏细辛对大鼠急性高眼压的缓解及视网膜神经节细胞的保护作用

目的:观察灯盏细辛（EBHM）调控丝裂原活化蛋白激酶（MAPK）信号通路对大鼠急性高眼压的影响及视网膜神经节细胞（RGC）的保护作用。方法:雄性Sprague-Dawley大鼠60只,采用随机数字表法分为对照组、模型组、EBHM低剂量组（A组）、EBHM中剂量组（B组）、EBHM高剂量组（C组）,每组均为12只;以左眼...     

大鼠视神经损伤视网膜病理改变的实验研究

目的研究现神经损伤早期视网膜的病理改变。方法采用大鼠球后视神经横断伤和钳夹伤模型,观察视网膜组织学及超微结构的改变。结果1）光镜：早期表现为神经纤维层血管扩张,损伤后7、14天可见散在的核染色质边聚。空化的节细胞。2）电镜：损伤后1天节细胞出现胞浆成分疏松,内质网扩张等变性样改变,横断伤3天及钳夹伤7天可见节细胞坏死,部分节细胞凋亡。结论视神经损伤导致节细胞出现迟发性死亡,提示早期治疗具有重要意义。     

兔视神经损伤后视网膜神经节细胞MDA/SOD的变化

目的:观察兔视神经损伤后视网膜神经节细胞(retinal ganglion cell,RGC)丙二醛(malondialdehyde,MDA)/超氧化物歧化酶(superoxide dismutase,SOD)的变化。方法:选用新西兰白兔32只,分为对照组和损伤组,损伤组再根据损伤后不同时间分为3,7,14d组,每组8只16眼。采用硫代戊巴比妥酸法和黄嘌呤氧化法分别测定视网膜MDA/SOD含量。结果:损伤实验组MDA含量分别为5.95±0.78,7.67±0.64和8.29±1.02μmol/g,均明显高于正常对照组(3.82±0.54μmol/g);实验各组SOD含量分别为510±44,415±29和398±36μkat/g,均明显低于正常对照组(727±52μkat/g)。结论:自由基代谢在兔视神经损伤后RGC凋亡中起着重要作用,MDA/SOD含量测定在RGC凋亡的研究中有一定的意义。     

大鼠神经损伤视网膜病理改变的实验研究

目的 研究神经损伤早期视网膜的病理改变。方法 采用大鼠球后视神经横断务和钳夹伤模型,观察视网膜组织学及超微铁改变。结果 １）光镜：早期表现为神经纤维层血管扩张,损伤后７、１４天可见散在的核染色质边聚。空化的节细胞。２）电镜：损伤后１天节细胞出现胞浆成分疏松,内质网扩张等变性样改变,横断作３天及钳夹伤７天可见节细胞坏死,部分节细胞凋亡。结论 视神经损伤导致节细胞出现迟发性死亡,提示早期治疗具有重要意     

兔视神经损伤后视网膜神经节细胞线粒体跨膜电位的变化观察

目的观察兔视神经损伤后视网膜神经节细胞（retinal ganglion cell,RGC）线粒体跨膜电位（mitochondrial transmembrane potentials,△ψm）的变化。方法选用新西兰白兔32只,分为对照组和损伤组,损伤组再根据损伤后不同时间分为3d、7d、14d组,共4组,每组8只兔16眼。采用Rh123染色,流式细胞术测定RGC△ψm。结果正常新西兰白兔RGC△ψm为3836.67±21.32。在损伤后3d、7d、14d,视网膜细胞△ψm分别为3055.41±7.74、1822.07±18.66、2617.38±7.37。各时间点的△ψm均明显低于正常对照组,且各时间点间△ψm差异有统计学意义（P〈0.05）。结论△ψm在RGC凋亡早期的观测中有一定的意义,并且能进行定量分析研究。     

Activation of autophagy in the retina after optic nerve crush injury in rats

AIM: To investigate the activation of autophagy in rat retina after optic nerve crush (ONC) and evaluate its relationship with apoptosis of retinal ganglion cells (RGCs). METHODS: The ONC model was established. Western blots were performed to investigate expression of p62, LC3 and Beclin-1. Transmission electron microscopy was performed to discover the autophagosomes in the retina after ONC. Immunohistochemistry was used to confirm the distribution of LC3. TUNEL was performed to confirm the relationship between autophagy and RGC apoptosis. RESULTS: p62/Beclin-1 ratio was declined shortly after ONC until to day 7 after ONC and then restored to a normal level at day 21. There was an opposite change in the LC3-II/LC3I ratio in the retina compared to the p62/Beclin-1 ratio. Increased autophagosomes were found after ONC using transmission electron microscopy, and most of the LC3-stained cells were colocalized with RGCs and Müller cells. More LC3-immunoreactive cells and apoptotic RGCs were found on day 7 following ONC. CONCLUSION: Possible activation of autophagy in RGCs after ONC; autophagy mainly occurred in RGCs and Müller cells, and the apoptosis of RGCs after ONC may be partly associated with autophagic activation.     

视神经挫伤后视网膜形态学和Bcl-2/Bax表达

目的 研究大鼠视神经夹挫伤后视网膜神经节细胞（RGC）形态学改变及Bcl-2／Bax蛋白表达的变化，为了解视神经损伤的病理机制提供一定的依据。方法 建立大鼠视神经夹挫伤动物模型，伤后1d、3d、5d、7d、9d、2周、4周处死，HE染色观察RGC的动态变化，免疫组化方法检测RGC表达Bcl-2及Bax的水平。结果 视神经伤后RGC数目严重下降，2周内RGC快速减少，2周以后缓慢减少；伤后Bcl-2及Bax表达随时间而有不同程度的增加，Bax对损伤的反应较Bcl-2稍晚，两者表达均呈现先升后降的趋势，并维持一定的时间。Bcl-2和Bax蛋白表达比与RGC存活数目有一定的相关性。结论 视神经损伤后RGC数目减少是其视功能下降的病理基础之一，Bcl-2和Bax在RGC死亡机制中起重要作用，Bcl-2／Bax比率与RGC的减少呈一定的相关性。     

大鼠视神经横断伤及夹挫伤后视网膜神经节细胞变化及与损伤时间关系

目的 观察大鼠视神经横断伤及夹挫伤后,视网膜神经节细胞(RGCs)形态学变化、区别及在不同时间的计数变化,探讨其与视神经损伤经过时间的关系,为视神经损伤的病理机制及损伤经过时间的推断提供一定的依据.方法 采用大鼠球后视神经横断伤/夹挫伤动物模型,在伤后不同时间处死动物并取材,HE 染色,光镜下观察RGCs的动态变化.结果 视神经损伤后RGCs数日均严重下降,2周内RGCs快速减少,3～7 d为RGCs快速减少期,2周以后缓慢减少;但横断伤组3 d以后各个时期RGCs计数下降幅度与夹挫伤组相比更明显.结论 视神经损伤导致了视网膜形态结构的变化,RGCs丢失的严重程度与损伤类型及时问呈相关性.     

Latanoprost protects rat retinal ganglion cells from apoptosis in vitro and in vivo

We investigated whether latanoprost has a direct anti-apoptotic effect in retinal ganglion cell (RGC) line and RGCs in the rat. RGC-5 cells were induced to undergo apoptosis by serum deprivation and exogenous glutamate. The level of cell death with or without latanoprost acid was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in the level of intracellular calcium ([Ca²⁺]i) were measured with fluo-4 fluorescence. The XTT assay revealed that latanoprost acid increased RGC-5 cell viability. Latanoprost acid significantly reduced caspase-3 positive cells and suppressed [Ca²⁺]i evoked by glutamate. U0126, a mitogen-activated protein/extracellular signal-regulated kinase 1 and 2 inhibitor, partially blocked the rescue effect of latnanoprost acid (p = 0.013). In vivo, rat RGCs were degenerated by optic nerve crush. After topical instillation of latanoprost for 7 days, RGCs labeled with fluorogold were significantly. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells. TUNEL-positive cells were significantly decreased in eyes with topically instilled latanoprost (p = 0.015). These data suggest that latanoprost has an neuroprotective ability in RGCs.     

Tumstatin肽对视网膜微血管内皮细胞迁移及P38MAPK蛋白表达的影响

目的:观察tumstatin肽对体外培养的视网膜微血管内皮细胞迁移及P38MAPK蛋白表达的影响,初步探讨tumstatin肽抗视网膜内皮细胞迁移的机制。方法:采用细胞划痕实验测定tumstatin肽(T8肽)对血管内皮生长因子(VEGF)诱导下RF/6A细胞(恒河猴视网膜微血管内皮细胞)迁移的影响;Western blotting检测T8肽对VEGF刺激后15,30,45,60min的RF/6A细胞P38MAPK蛋白水平的变化。结果:Tumstatin肽对RF/6A细胞迁移具有抑制作用,且可抑制VEGF对RF/6A细胞的促迁移作用,呈剂量依赖性。正常情况下,RF/6A细胞无P38MAPK蛋白的表达,但VEGF可诱导其表达P38MAPK蛋白,而tumstatin可抑制VEGF诱导的RF/6A细胞P38MAPK蛋白的表达(加入20mg/LT8肽30,45,60min时蛋白表达受到显著抑制,差异有显著性意义,P<0.01)。结论:Tumstatin抑制视网膜微血管内皮细胞的迁移,其作用可能与P38MAPK通路有关。     

The effect of ginkgo biloba on the rat retinal ganglion cell survival in the optic nerve crush model

Purpose: To investigate the effect of ginkgo biloba on the retinal ganglion cell survival in a rat optic nerve crush model. Methods: Twenty‐four Sprague–Dawley rats were divided randomly into a study group of 12 animals receiving intraperitoneal injections of ginkgo biloba and a control group of 12 animals receiving intraperitoneal saline injections. All injections were performed 1 hr before the optic nerve crush and daily afterwards. For each animal, the right optic nerve was crushed closely behind the globe for 60 seconds using a microclip with 40 g power. The left optic nerve was kept intact. At 23 days after the optic nerve crush, the retinal ganglion cells were labelled retrogradely by injecting 3% fluorogold into both sides of the superior colliculus of the brain. At 4 weeks after the optic nerve crush, the animals were killed. Photographs taken from retinal flat mounts were assessed for the number and density of the retinal ganglion cells. Results: The survival rate, defined as the ratio of the retinal ganglion cell density in the right eye with the optic nerve crush divided by the retinal ganglion cell density in left eye without an optic nerve trauma, was significantly (p = 0.035) higher in the study group with ginkgo biloba than in the control group (60.0 ± 6.0% versus 53.5 ± 8.0%). Conclusion: The results suggest that intraperitoneal injections of a ginkgo biloba extract given prior to and daily after an experimental and standardized optic nerve crush in rats were associated with a higher survival rate of retinal ganglion cells.     

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。     

Citicoline and lithium rescue retinal ganglion cells following partial optic nerve crush in the rat

Citicoline and lithium (Li(-)) have been shown to support retinal ganglion cell (RGC) survival and axon regeneration in vitro. Optic nerve crush (ONC) is a model of both brain axonal injury and certain aspects of the glaucomatous degeneration of RGC. We have used this model to quantify protection offered to RGC by these drugs and to determine whether their effects are mediated by enhanced expression of the antiapoptotic protein Bcl-2. Adult rats (6-12 per group) were subjected to ONC accompanied by a contralateral sham operation. Animals were treated intraperitoneally with either vehicle, citicoline sodium (1g/kg daily for up to 7 days and 300 mg/kg daily afterwards), lithium chloride (30 mg/kg daily), or both drugs combined. Fluorogold was injected bilaterally into superior colliculi 1, 5 or 19 days after ONC. Labeled cells were counted under a fluorescence microscope 2 days after tracer injection. In a separate set of experiments the effects of treatments on expression of Bcl-2 in retinas were evaluated by immunohistochemistry. In vehicle-treated animals there was a progressive decrease of RGC density after crush. This decrease was attenuated in citicoline-treated animals 1 week and 3 weeks after the crush. In the lithium-treated group protection was even more pronounced. In animals treated with both drugs RGC protection was similar to that achieved by lithium alone. Bcl-2 immunoreactivity was seen predominantly in retinal ganglion cells. Its increase was recorded in the lithium and citicoline group as well as in animals treated with the combination of both drugs. Both citicoline and lithium protect RGC and their axons in vivo against delayed degeneration triggered by the ONC. Retinoprotective action of both drugs may involve an increase in Bcl-2 expression.     

甲钴胺对钳夹伤大鼠视神经的保护作用

目的 :通过动物实验观察视神经钳夹伤后甲钴胺对其的保护作用。方法 :取成年S D大鼠 38只 ,均成功制成视神经钳夹伤的模型 ,随机将其分为 7组 :损伤后 1个月对照组、损伤后 2个月对照组各 6只 ;VitB12 组、甲钴胺组、甲钴胺加倍剂量组各 6只 ,分别采用VitB12 0 .0 2ml(10 μg)、甲钴胺 0 .0 2ml(10 μg)、甲钴胺 0 .0 4ml(2 0 μg)于损伤当时及以后隔日行臀大肌注射 ,1个月后取材 ;甲钴胺 2个月组、甲钴胺加倍剂量 2个月组各 4只 ,治疗至 2个月后取材。观察大鼠视神经轴突和视网膜神经节细胞 (RGC)的形态及数目变化。收稿日期 :2 0 0 3 -12 -0 6;修回日期 :2 0 0 4-0 4-0 3作者简介 :孔祥梅 ( 1978-) ,女 ,医学硕士 ,研究方向 :青光眼。通信作者 :孙兴怀 (E -mail:xhsun @shmu .edu .cn)结果 :从轴突、RGC形态看 ,VitB12 组较损伤对照组形态变化不大 ,而甲钴胺各治疗组异形改变较少。VitB12 组轴突及RGC数目分别为 35 4 .6 7± 4 4 .72和 4 4 .0 0± 8.12 ,与损伤后 1个月对照组相比未见明显增多 (P分别为 0 .2 32、0 .170 ) ;甲钴胺组轴突及RGC数目分别为 4 5 2 .17± 83.5 8和 5 8.0 0± 6 .38,与损伤后 1个月对照组相比显著增多 (P分别为 0 .0 10、0 .0 0 3) ,与VitB12 组相比 ,数目增多差异也有显著