bcl-X_L抗视网膜光感受器细胞凋亡作用的实验研究

目的　评价抗凋亡基因bcl XL 对视网膜光感受器细胞的抗凋亡作用。方法　首先建立谷氨酸损伤的SD大鼠视网膜光感受器细胞凋亡模型。将体外培养的光感受器细胞分为A组 (正常对照组 )、B组 (谷氨酸组 )及C组 (rAd gfp bcl XL 转染 +谷氨酸组 ) ;其中C组在加入谷氨酸前 4 8h用滴度为 6 5×l0 12 pfu/mL的重组腺病毒rAd gfp bcl XL 转染光感受器细胞 ;荧光显微镜下观察光感受器细胞中的绿色荧光蛋白表达情况 ;用免疫组化法分析rAd gfp bcl XL 转染细胞和未转染细胞Bcl XL蛋白水平 ;DNA琼脂糖凝胶电泳以评判 3个组神经元凋亡发生与否及凋亡程度 ;采用Hoechst332 5 8染色做正常核和凋亡核的形态学检测。结果　免疫组化检测结果表明转染细胞与未转染细胞的Bcl XL 蛋白表达水平有差异 ,DNA电泳分析发现B组呈典型的DNA“梯度”条带 ,而A组和C组几乎无DNA“梯度”条带 ,核形态学检测结果亦证实转染组较未转染组的核有明显差异。结论重组腺病毒介导转染的bcl XL 对体外培养的视网膜光感受器细胞有抗凋亡作用 ,提高视网膜光感受器细胞bcl XL 的表达水平可能为视网膜变性疾病提供潜在有效的治疗方法。     

抗凋亡基因bcl-XL转染视网膜感光细胞的研究

目的 :探讨介导抗凋亡基因bcl-XL 转染视网膜感光细胞的有效方法。方法 :采用视网膜神经胶质细胞条件培养液体外培养的视网膜感光细胞 ;用脂质体LIPOFECTAMINETM2 0 0 0介导 pEGFP -C3 -bcl-XL 转染至感光细胞 ;采用滴度为 6 5× 10 12 pfu/L的重组腺病毒rAd -gfp -bcl-XL 转染感光细胞 ;在荧光显微镜下观察感光细胞中的绿色荧光蛋白表达情况来比较脂质体、重组腺病毒转染法的转染率 ;用免疫组化比较这两种方法转染的细胞中Bcl-XL蛋白水平。结果 :脂质体、重组腺病毒转染法的转染率分别为 0 1%、 60 % ,免疫组化示重组腺病毒转染的细胞的Bcl -XL 蛋白水平较脂质体转染的细胞高。结论 :在视网膜感光细胞的基因转染中 ,腺病毒介导的目的基因bcl-XL 的转染率及表达明显高过脂质体法 ,重组腺病毒介导的基因转染法是视网膜变性性疾病基因治疗的一种较理想方法。     

重组腺病毒介导的gfp-bcl-X_L对视网膜变性鼠光感受器细胞保护作用的研究

目的　观察重组腺病毒rAd gfp bcl XL治疗视网膜变性 (retinaldegeneration ,RD)鼠的疗效。方法　 40只RD鼠随机分成A、B组 ,每组 2 0只 ,右眼为治疗眼 ,左眼为对照眼。A、B组治疗眼分别于RD鼠生后 10d及 2 2d视网膜下腔注射含绿色荧光蛋白基因gfp和抗凋亡基因bcl XL 的重组腺病毒颗粒 ;术后 15、30d摘除治疗眼和对照眼行冰冻切片、透射电镜下观察、石蜡切片、HE染色并提取视网膜总RNA ;行逆转录聚合酶链反应分析bcl XL 基因的mRNA表达水平 ;免疫组化分析治疗眼和对照眼的Bcl XL 蛋白水平。结果　治疗眼视网膜有较广泛的绿色荧光蛋白表达 ,说明重组腺病毒成功的将bcl XL 转染至视网膜光感受器细胞 ,且有bcl XL 表达 ;术后 30d ,A组鼠治疗眼和对照眼bcl XL的mRNA表达水平及Bcl XL 蛋白含量有明显差异 ;透射电镜下可见治疗眼的内节段较对照眼完好 ;HE染色示治疗眼视网膜光感受器细胞层较对照眼厚。而B组鼠治疗眼和对照眼的视网膜光感受器细胞层的厚度无明显差异。结论　注射至早期RD鼠视网膜下腔中的rAd gfp bcl XL 能有效转染光感受器细胞并发挥抗凋亡作用 ,但对晚期RD鼠的疗效不明显。     

重组腺病毒介导的gfp-bcl-XL对视网膜变性鼠光感受器细胞保护作用的研究

目的 观察重组腺病毒rAd-gfp-bcl-XL治疗视网膜变性(retinal degeneration,RD)鼠的疗效。方法 40只RD鼠随机分成A、B组,每组20只,右眼为治疗眼,左眼为对照眼。A、B组治疗眼分别于RD鼠生后10d及22d视网膜下腔注射含绿色荧光蛋白基因gfp和抗凋亡基因bcl-XL的重组腺病毒颗粒;术后15、30d摘除治疗眼和对照眼行冰冻切片、透射电镜下观察、石蜡切片、HE染色并提取视网膜总RNA;行逆转录聚合酶链反应分析bcl-XL基因的mRNA表达水平;免疫组化分析治疗眼和对照眼的Bcl-XL蛋白水平。结果 治疗眼视网膜有较广泛的绿色荧光蛋白表达,说明重组腺病毒成功的将bcl-XL转染至视网膜光感受器细胞,且有bcl-XL表达;术后30d,A组鼠治疗眼和对照眼bcl-XL的mRNA表达水平及Bcl-XL蛋白含量有明显差异;透射电镜下可见治疗眼的内节段较对照眼完好;HE染色示治疗眼视网膜光感受器细胞层较对照眼厚。而B组鼠治疗眼和对照眼的视网膜光感受器细胞层的厚度无明显差异。结论 注射至早期RD鼠视网膜下腔中的rAd-gfp-bcl-XL能有效转染光感受器细胞并发挥抗凋亡作用,但对晚期RD鼠的疗效不明显。     

挫伤性视网膜病变中光感受器细胞凋亡与氧化损伤的实验研究

目的　探讨挫伤性视网膜病变中视网膜损伤的凋亡机制及诱因。方法　以 3J能量自由落体的方式制作兔眼挫伤性视网膜病变模型 ,通过光、电镜及TUNEL染色法观察视网膜病变及细胞凋亡情况 ,测定视网膜匀浆中MDA含量与SOD活性的变化 ,并行统计学分析。结果　以 3J能量挫伤后的视网膜病变中存在着光感受器细胞凋亡现象。随挫伤时间推移 ,视网膜MDA的含量逐渐升高 (P <0 .0 1) ,SOD活性在挫伤后早期反射性升高 (P <0 .0 1) ,3d时明显下降 (P <0 .0 1) ,且与凋亡细胞出现的时间一致。结论　细胞凋亡是挫伤性视网膜病变的一个重要机制。活性氧自由基是挫伤性视网膜病变中光感受器细胞凋亡的重要诱因。     

小鼠实验性视网膜脱离后光感受器细胞的凋亡

目的 研究小鼠实验性视网膜脱离后光感受器细胞的凋亡情况。 方法 将成年C57Bl/6J小鼠36只分为2组：实验组小鼠18只左眼视网膜下注射1.4%透明质酸钠造成视网膜脱离，对照组小鼠18只左眼仅作巩膜穿刺。分别于手术后1、3、7和28 d摘除眼球，视网膜切片进行组织化学、免疫荧光染色，共聚焦显微镜检查。抗视锥和抗视杆细胞的抗体分别标记视锥和视杆细胞，dUTP缺口末段标记法(TUNEL)标记凋亡细胞。通过计数存活和凋亡的视锥和视杆细胞来定量光感受器细胞的凋亡和细胞丢失。 结果 凋亡细胞只存在于脱离部分视网膜的外核层，凋亡细胞在视网膜脱离后1 d即可检测得到，3 d时达到高峰，7 d后陡然减少。视网膜脱离后视杆和视锥细胞的死亡呈现同样的时程。 结论 凋亡是视网膜脱离后光感受器细胞死亡的主要病理改变。 (中华眼底病杂志, 2006, 22: 124-127)     

应重视视网膜光化学损伤光感受器细胞凋亡的研究

视网膜退行性变性包括年龄相关性黄斑变性、视网膜色素变性等,其重要病理特征为进行性视网膜光感受器细胞凋亡,目前尚无有效的治疗方法.近年来许多流行病学调查和实验研究表明,可见光可促使视网膜退行性变性恶化,因此视网膜光化学损伤和光感受器细胞凋亡的研究成为重要的研究方向.(中华眼科杂志,2009,45:196-198)     

视网膜脱离视细胞凋亡及其抗凋亡治疗研究进展

视网膜脱离是主要的致盲眼病之一,其发生机制纷繁复杂.目前研究认为光感受器等视细胞的凋亡在视网膜脱离预后中起重要作用.视网膜脱离视细胞凋亡的机制逐步被阐明,相关的光感受器细胞抗凋亡治疗研究也逐渐成为治疗视网膜脱离的热点.光感受器细胞的抗凋亡治疗结合现代视网膜脱离手术将是未来治疗视网膜脱离的理想模式.     

细胞凋亡与视网膜变性

细胞亡是细胞的一种特殊方式，不同于细胞死亡，它既可以是生理性的，也可以是病理的性，文中阐述了细胞凋亡的基本概念，分子生物学特点，凋亡与坏死的区别，及其在视网膜退行性病变听原关联。     

Structural and functional protection of photoreceptors from MNU-induced retinal degeneration by the X-linked inhibitor of apoptosis

PURPOSE: To evaluate the neuroprotective effects of adenoassociated virus delivery of XIAP in N-methyl-N-nitrosourea (MNU)-induced retinal degeneration in Sprague-Dawley rats. METHODS: Sprague-Dawley rats were injected subretinally with recombinant adenoassociated virus (rAAV) encoding either XIAP or green fluorescent protein (GFP; injection control). Six weeks after injection, the animals received an intraperitoneal injection of MNU, a DNA methylating agent, at a dose of 60 mg/kg. Electroretinograms (ERGs) were recorded at 0, 24, 48 and 72 hours and 1 week after MNU. The rats were killed after the ERG was performed and were perfused with 4% paraformaldehyde. Eyes were then enucleated and embedded for cryosectioning. Eye sections were analyzed by TUNEL and histologic techniques. Real-time PCR and Western analysis were performed to confirm the overexpression of XIAP in injected eyes. RESULTS: Real-time PCR and Western analysis confirmed the overexpression of XIAP in virus-injected eyes in comparison to uninjected control eyes. At 24 hours after MNU injection, fewer cells had undergone apoptosis in the XIAP-treated eyes in comparison with GFP-injected or uninjected eyes. Hematoxylin and eosin staining revealed that the uninjected and GFP-injected photoreceptors were destroyed by 72 hours after injection of MNU, whereas the AAV-XIAP-injected eyes showed structural protection of the photoreceptors at all time points throughout the 1-week sampling period. ERGs showed functional protection up to 1 week after MNU injection in the AAV-XIAP-injected eye, whereas no response was observed in the control eye. CONCLUSIONS: The results suggest that XIAP is protective against this potent chemotoxic agent and holds promise as a therapeutic agent in gene therapy approaches to treating retinitis pigmentosa.     

Unoprostone isopropyl rescues retinal progenitor cells from apoptosis in vitro

PURPOSE: Unoprostone isopropyl is an ocular hypotensive that was originally produced as a prostaglandin F2alpha analogue and is eventually recognized as a synthetic docosanoid. The compound is recently suggested to have potent neuroprotective ability in the retina. The purpose of this study is to test whether and how the biologically active metabolites of unoprostone isopropyl rescue retinal neuro-glial progenitor cells from apoptosis. METHODS: R28 cells were deprived of serum for 24 hr with or without varying concentrations of unoprostone metabolite M1 or M2 or vehicle in the presence or absence of specific inhibitors against several types of signal transduction proteins. Immunocytochemistry against activated caspase-3 with Hoechst nuclear staining was performed. RESULTS: Up to 15%of R28 cells became pyknotic and activated caspase-3 immunoreactive after 24-hr serum withdrawal. M1, but not M2, significantly reduced apoptotic cells in a dose-dependent fashion with a maximal effect at 100 microM (p < .0001). LY294002, the phosphatidylinositol 3-OH kinase (PI3K) inhibitor, and KT5823, the protein kinase G (PKG) inhibitor, reversed the antiapoptotic effect of M1. CONCLUSIONS: The unoprostone metabolite M1 protects retinal neuro-glial progenitor R28 cells from apoptosis induced by serum deprivation via the PI3K and PKG pathways.     

Fine structure of the lamprey photoreceptors and retinal pigment epithelium (Petromyzon marinus L.).

The retinal photoreceptor cells and pigment epithelium of the lamprey, Petromyzon marinus have been examined with the electron microscope.The pigment epithelial cells in this avascular retina are characterized by surface specializations in the form of apical microvilli and basal infoldings. These cells contain myriad myeloid bodies, some phagosomes and residual bodies, as well as sparse, apically positioned melanin granules.Two types of photoreceptor cells are described in the retina of this animal. The long receptors have conical outer segments, slender elongated myoid regions and their nuclei are positioned in the outer portion of the outer nuclear layer near the external limiting membrane. The pyramid-shaped synaptic terminals of these cells are found deep in the outer plexiform layer, are less electron dense than those of the short receptors and their synaptic vesicles appear to be less densely packed than in the long receptor terminals. The short receptors have slender, elongated outer segments, their nuclei are positioned deeper in the outer nuclear layer, while the spherical synaptic terminals are located in the scleral region of the outer plexiform layer. The outer segments of both cell types however, show similar cone-like characteristics at the ultrastructural level, being composed of membrane-bound stacks of double-membrane discs, some of which are in continuity with the extra-cellular space. The outer segment of both types of receptors is joined to the inner segment through a slender ciliary stalk and is surrounded by calycal processes.Autoradiographic analysis shows that protein renewal in the photoreceptor outer segments of both long and short receptors takes place in a similar diffuse manner, giving rise are to the suggestion that both photoreceptor types in the retina of Petromyzon marinus cones.     

Genetic ablation of cone photoreceptors eliminates retinal folds in the retinal degeneration 7 (rd7) mouse

PURPOSE: Folds or pseudorosettes in the outer retina are commonly observed in animals with genetic, viral, or chemically induced retinal degeneration. This study examined the effect of genetic ablation of cone photoreceptors on the production of retinal folds in two mouse retinopathy models. One is the rd7/rd7 retina, a model of enhanced S-cone syndrome, Goldman-Favre syndrome, and clumped pigmentary retinopathy that is also associated with an approximately twofold excess of S-cones. The other is postnatal day (P)0 N-methyl-N-nitrosourea (MNU) treatment. METHODS: A transgene that directs cone-specific expression of the diphtheria toxin A chain was used to ablate cone photoreceptors. Retinal folds, numbers of photoreceptors, and numbers of cones were quantified in retina flatmounts or transverse sections, and photoreceptor apoptosis was quantified by immunostaining for activated caspase 3. RESULTS: Cone ablation by the cone-DTA transgene eliminated folds in the rd7/rd7 retina, whereas chemical ablation of up to 30% of rods (by exposure to MNU at P13) had little or no effect on folds in the rd7/rd7 retina. Cone ablation by the cone-DTA transgene had no effect on retinal folds produced by P0 MNU treatment or on the progressive loss of rod photoreceptors in the rd7/rd7 retina. CONCLUSIONS: Despite their relatively low abundance, cones play a critical role in retinal folding in the rd7/rd7 retina. The relevant molecular and cellular mechanisms remain to be determined.     

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.     

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

Background

To investigate whether tafluprost, which is a prostaglandin-related compound and an anti-glaucoma drug, has a direct anti-apoptotic effect in cultured retinal ganglion cells (RGCs) and rat RGCs in retinas with optic nerve crush (ONC).

Methods

RGC-5 cells were induced to undergo apoptosis by a serum deprivation and by exogenous glutamate. The level of cell death with or without tafluprost was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in intracellular calcium ([Ca²⁺]i) levels were measured with fluo-4 fluorescence. Rat RGCs were degenerated by ONC. After topical instillation of tafluprost for 7 and 14 days, the numbers of retrograde-labeled RGCs were counted. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells.

Results

Tafluprost dose-dependently promoted RGC-5 cell viability with an optimum concentration of 3?μM (p?=?0.006). Tafluprost significantly reduced caspase-3-positive cells and suppressed [Ca⁺²]i evoked by exogenous glutamate. The cGMP-dependent protein kinase inhibitor and KT-5823 partially blocked the rescue effect of tafluprost (p?=?0.002). The survival rate of RGCs significantly increased in eyes treated with tafluprost (p?=?0.01), and the prevalence of TUNEL-positive cells was significantly decreased 14 days after ONC (p?<?0.001).

Conclusions

These data suggest that tafluprost has an anti-apoptotic effect in RGCs.     

Immunoferritin localization of actin in retinal photoreceptors

The contractile protein actin was recently localized to the distal portion of the connecting cilium in frog photoreceptors (Chaitin et al J Cell Biol 99:239-247, 1984). This is the site where the ciliary plasma membrane evaginates to form new outer segment disks (Steinberg et al J Comp Neurol 190: 501-518, 1980). In the present study, aldehyde fixed mammalian retinas were embedded in Lowicryl K4M, and thin tissue sections were reacted with antiactin antibodies using indirect immunoferritin labeling. Utilizing this technique, actin has been localized to the distal cilium in rat, cow, monkey, and human photoreceptors. These results provide additional evidence that an actin mediated contractile mechanism may regulate outer segment disk morphogenesis in vertebrate photoreceptors. As previously noted in frog retina, antiactin also labeled the bundle of filaments within photoreceptor calycal processes, and this label extended into the inner segment, subjacent to the plasma membrane. Within the inner segment, however, the striated rootlet was unlabeled.