视网膜感光细胞的凋亡与保护

许多视网膜变性疾病,尽管其发病机制和临床特点不同,但最终均以感光细胞的凋亡而引起不可逆的视力丧失。本文阐述了细胞凋亡的共同通路：Ｃａｓｐａｓｅ激活的酶促反应;同时分析了感光细胞凋亡与ＡＰ－１因子的关系,并进一步对感光细胞变性特点及ｂＦＧＦ、ＤＨＡ的保护作用一综述。     

小胶质细胞活化与rd小鼠遗传性视网膜变性

目的研究小胶质细胞活化与rd小鼠遗传性视网膜变性的关系。方法对出生后8、10、12、14、16及18d的rd小鼠及对照小鼠视网膜进行感光细胞凋亡TUNEL法检测及形态计量学分析。CD11b免疫组织化学染色标记视网膜小胶质细胞。结果rd小鼠出生后10d视网膜感光细胞层开始出现TUNEL染色阳性细胞,第16d达到高峰。视网膜小胶质细胞在rd小鼠出生后10d开始活化,第14d达到高峰。小胶质细胞向感光细胞层的迁移与感光细胞凋亡之间存在紧密的时间和空间关系。结论rd小鼠视网膜变性以感光细胞凋亡为主。小胶质细胞活化可能在视网膜变性过程中发挥重要作用。     

NADPH氧化酶抑制剂对遗传性视网膜色素变性感光细胞凋亡的抑制作用

背景 我们先前的研究表明,rd小鼠遗传性视网膜色素变性（RP）过程中小胶质细胞活化与感光细胞的凋亡密切相关.研究显示,小胶质细胞中烟酰胺二磷酸腺苷（NADPH）氧化酶的活化在小胶质细胞活化及神经元损伤中发挥重要作用,但NADPH氧化酶在RP过程中作用机制及其抑制剂的作用有待探讨.目的 探讨rd小鼠发生RP过程中NADPH氧化酶产生活性氧簇（ROS）的活化反应及其抑制剂对感光细胞的保护作用. 方法 按抛掷硬币法将60只SPF级rd小鼠随机分为香荚兰乙酮注射组和PBS对照组,香荚兰乙酮注射组小鼠于出生后9d（P9）腹腔内注射NADPH氧化酶抑制剂香荚兰乙酮10 mg/kg（0.01 ml/kg）,每日1次,连续5d（至P13）;PBS对照组rd小鼠以同样方式注射等容量的PBS;C57 BL/6N小鼠10只不注射任何药物作为rd小鼠的野生对照鼠.各组小鼠于出生后14 d（P14）处死并制备视网膜冰冻切片,采用二氢乙锭（DHE）荧光染色法检测3个组小鼠视网膜中ROS的表达;采用实时定量PCR（real-time PCR）法测定2个组rd小鼠视网膜感光细胞中视紫红质mRNA的定量表达;采用苏木精-伊红染色法检查2个组rd小鼠视网膜外核层厚度.结果 DHE荧光染色表明,小鼠视网膜中ROS表达呈红色荧光,注药组小鼠视网膜外核层中ROS的红色荧光明显强于C57BL/6N野生鼠,但明显弱于PBS对照组.Real-time PCR检测表明,香荚兰乙酮注射组小鼠感光细胞中视紫红质mRNA相对表达量为4.21 ±0.33,明显低于PBS对照组的0.93±0.24,差异有统计学意义（t=2.360,P=0.000）;香荚兰乙酮注射组小鼠视网膜外核层厚度为（35.95±1.63） μm,明显厚于PBS对照组的（23.17±1.38） μm,差异有统计学意义（t=3.850,P=0.016）.结论 在rd小鼠视网膜感光细胞变性过程中,NADPH氧化酶生成ROS的活化反应明显增强;香荚兰乙酮能够延缓rd小鼠感光细胞的凋亡过程.     

rd11小鼠视锥细胞变性过程中视蛋白的变化特点

背景视网膜变性11（rd11）小鼠是近年来新发现的一种自发突变的视网膜变性小鼠。研究证实,rd11小鼠出生后随着鼠龄的增长出现快速的光感受器变性,且视杆细胞变性早于视锥细胞变性,但对于视网膜不同区域视锥细胞变性的特点还不十分清楚。目的应用视网膜铺片免疫荧光染色技术观察不同鼠龄rd11小鼠M-视蛋白和S-视蛋白在视网膜的表达分布及变化特点,为相关疾病的基因治疗研究提供实验依据。方法取出生后14、28、42d的rd11小鼠各5只,制备视网膜铺片,采用免疫荧光组织化学法分别标记小鼠视网膜后极部颞上、颞下、鼻上和鼻下象限M-视蛋白和S-视蛋白的表达,观察随rd11小鼠鼠龄的变化视网膜各区域M-视蛋白和S-视蛋白的荧光形态和密度,并与相应鼠龄的C57BL／6J小鼠进行比较。结果出生14d的rd11小鼠视网膜M-视蛋白和S-视蛋白的红色荧光形态和密度与C57BL／6J小鼠接近,但出生28d的rd11小鼠视网膜后极部颞上、颞下、鼻上、鼻下4个区域M-视蛋白和S-视蛋白表达密度均明显降低,荧光形态由纺锤形逐渐变为点状,出生42d的rd11小鼠视网膜部分区域M-视蛋白和S-视蛋白表达消失。出生28d的rd11小鼠视网膜后极部颞上、颞下、鼻上、鼻下区域M-视蛋白的表达密度分别为（414±32）、（300±8）、（324±22）和（250±20）个／0．037mm^2,明显低于同龄C57BL／6J小鼠的（484±21）、（442±19）、（459±34）和（436±12）个／0．037mm^2,差异均有统计学意义（t=4．114、15．225、7．505、17．990,均P〈0．05）;上述4个区域S-视蛋白的表达密度下降更明显,分别为（8±4）、（175±16）、（74±13）、（315±20）个／0．037mm^2,明显低于C57BL／6J小鼠的（73±16）、（436±30）、（393±30）和（480±19）个／0．037mm^2,差异均有统计学意义（t=8．555、17．076、21．637、13．498,均P〈0．05）。结论rd11小鼠视锥细胞中的M-视蛋白和S-视蛋白均随着鼠龄的增长而急剧减少,以S-视蛋白更为明显。随着鼠龄的增长,rd11小鼠M-视蛋白变性从视神经周围区向鼻下方,再向颞上方逐渐进展,而S-视蛋白变性由颞上方向鼻下方逐渐进展。     

视网膜感光细胞凋亡机制的研究进展

视网膜变性是导致眼睛永久失明的主要原因之一,其特征是视网膜感光细胞的丧失,而感光细胞的丧失主要通过凋亡途径。近年来,虽然感光细胞的凋亡机制研究已经引起了重视,但目前仍缺乏有效的方式来抑制感光细胞凋亡。因此,本文将从凋亡的进程、凋亡发生过程中的关键分子、凋亡通路以及凋亡和自噬的联系等方面对其进行综述,旨在进一步阐明感光细胞凋亡的分子机制。     

NADPH氧化酶在rd小鼠遗传性视网膜变性中的活化表达

背景研究表明,小胶质细胞中烟酰胺二磷酸腺苷（NADPH）氧化酶的活化在中枢神经系统神经元损伤及神经变性疾病中发挥重要作用。已有研究证实NADPH氧化酶在rd小鼠视锥细胞退行性改变中的作用,但关于其在rd小鼠视网膜变性早期视杆细胞病变过程中的作用研究较少。目的研究NADPH氧化酶在rd小鼠感光细胞凋亡过程中的活化表达,探讨其在遗传性视网膜变性中的致病作用。方法取出生后8、10、12、14、16、18d的rd小鼠各18只,过量麻醉法处死后提取视网膜总RNA和总蛋白,并制备视网膜切片,分别用实时荧光定量PCR及Western blot法测定在rd小鼠感光细胞凋亡过程中视网膜中NADPH氧化酶亚单位gp91phox mRNA及蛋白的定量表达变化;采用免疫组织化学及gp91phox和CD11b免疫荧光双染法确定gp91phox在不同鼠龄rd小鼠视网膜中的表达及定位,并与其近交系C57BL／6N小鼠进行比较。结果实时荧光定量PCR研究证实,C57BL／6N小鼠视网膜中未见gp91phox mRNA的表达,而生后8d的rd小鼠视网膜中即有少量gp91phox mRNA的表达,随着鼠龄的增加,gp91phox mRNA表达量（gp91phox mRNA／β-actin）逐渐增加,生后14d达到高峰。不同鼠龄rd小鼠视网膜中gp91phox mRNA表达量差异有统计学意义（F=17．81,P=0．00）;生后10、12、14、16、18d的Td小鼠视网膜中gp91phox mRNA表达量均明显高于出生后8d小鼠,差异均有统计学意义（均P〈0．05）,以出生后14d表达量最高,为1．136±0．370。随着小鼠鼠龄的增加,视网膜中gp91phox蛋白表达量（A值）与其基因表达变化趋势一致,不同鼠龄及C57BL／6N对照小鼠间视网膜中gp91phox蛋白表达的差异有统计学意义（F=354．00,P〈0．01）,不同鼠龄rd小鼠出生后视网膜中gp91phox蛋白表达量均明显高于C57BL／6N对照小鼠,差异均有统计学意义（均P〈0．05）。免疫组织化学法检测表明,rd小鼠出生后10d视网膜内层gp91phox阳性细胞开始增多,出生后14d达到高峰,外核层也可见到gp91phox阳性细胞。免疫荧光双染结果显示,gp91phox与小胶质细胞标志物CD11b共表达,呈现橙色荧光。结论NADPH氧化酶在rd小鼠视网膜中的表达随着鼠龄的增长而增多,其表达与小胶质细胞活化及感光细胞的凋亡相平行,提示NADPH氧化酶可能在小胶质细胞活化导致的感光细胞凋亡中发挥致病作用。     

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。     

甲基苯丙胺对CD-1小鼠视网膜毒性作用的初步研究

目的 探讨甲基苯丙胺对CD-1小鼠视网膜的毒性作用.方法 12只CD-1小鼠腹腔内单次注射大剂量(40mg/kg)甲基苯丙胺.免疫组织化学法观察给药后1 d及7 d视网膜组织毒性反应.GFAP免疫标记大胶质细胞;CD11b免疫标记小胶质细胞;酪氨酸羟化酶(TH)标记多巴胺细胞及神经末梢改变.结果 全部CD-1小鼠耐受该给药方案.给药后1 d及7 d均发现视网膜大胶质细胞及小胶质细胞增生,活化.酪氨酸羟化酶变化不明显.结论 全身单次大剂量注射甲基苯丙胺后可能对CD-1小鼠视网膜产生一定毒性作用.     

中药复方制剂对rds小鼠感光细胞凋亡的干预作用研究

目的观察中药复方制剂对先天性视网膜变性小鼠视网膜感光细胞凋亡的影响。方法以黄芪等药组成复方制剂1。对先天性视网膜变性动物模型rds小鼠,采用原位末端转移酶标记(TUNEL)方法及组织病理学技术,观察复方制剂1作用后,小鼠视网膜感光细胞数量及感光细胞凋亡的变化。结果仔鼠2周时,中药组小鼠视网膜感光细胞核数与对照组相比无明显差别,两组感光细胞凋亡率分别为1.6%及6.5%,组间差异有显著性(P<0.01);4周时中药组感光细胞核数目较对照组多,差异有显著统计学意义(P<0.01),两组感光细胞凋亡率分别为3.3%及8.5%,组间差异有显著统计学意义(P<0.01)。结论中药复方制剂1可以延缓rds小鼠视网膜色素变性过程中感光细胞凋亡的发展。     

Netrin-1对糖尿病视网膜病变大鼠的保护作用

目的　探讨Netrin-1对糖尿病视网膜病变（diabetic retinopathy,DR）大鼠的保护作用。方法　通过腹腔注射链脲佐菌素诱导DR大鼠模型,使用生理盐水作为对照。为了研究Netrin-1基因沉默对DR的影响,构建si-NC和si-Netrin-1大鼠模型,并随机分为4组:A组（si-NC大鼠）、B组（si-Netrin-1大鼠）、C组（si-NC大鼠+DR造模）、D组（si-Netrin-1大鼠+DR造模）。为了研究外源性Netrin-1对DR的影响,将WT大鼠随机分为3组:对照组、DR组、Netrin-1组。造模后3个月,HE染色检测各组大鼠视网膜组织各层厚度,免疫荧光染色检测Iba1表达情况,实时荧光定量PCR检测各炎症因子表达水平。结果　与C组相比,D组大鼠视网膜组织中内核层、外核层厚度降低,差异均有统计学意义（均为 P<0.05）;Iba1及IL-1β、IL-6、IL-12、TNF-α表达升高,差异均有统计学意义（均为P<0.05）。与DR组相比,Netrin-1组内核层、外核层厚度升高,差异均有统计学意义（均为 P<0.05）;Iba1及各炎症因子表达水平降低,差异均有统计学意义（均为P<0.05）。结论　Netrin-1基因沉默可加重糖尿病大鼠视网膜损伤和炎症,外源性Netrin-1则具有保护作用。     

Genetic modification of retinal degeneration in tubby mice

Mice that carry the recessive mutation tub develop neurosensory defects including retinal and cochlear degeneration, as well as maturity-onset obesity associated with insulin resistance. The biological function of the gene and the mechanism by which it induces its phenotypes are still unclear. In order to elucidate the pathways through which tub functions, in the current study, QTL modifiers were identified in an F2 intercross between (C57BL/6J- tub/tub and AKR/J-+/+) F1 hybrids (AKR intercross). The thickness of the outer nuclear layer of the retina and the number of photoreceptor nuclei were assessed in F2 mice homozygous for the tub mutation. A genome-wide scan revealed a significant linkage on chromosome 11 (named motr1) and two suggestive linkages on chromosomes 2 and 8. Interestingly, the same chromosome 2 region identified for the hearing modifier of tubby, the moth1 locus, showed a peak lod score of 2.3 for protection from retinal degeneration. This result suggests that the gene responsible for the QTL on chromosome 2 might be involved in a common pathway through which retinal and cochlear degeneration are induced in tubby mice.     

Adenoviral-mediated gene transfer to retinal explants during development and degeneration

Naturally occurring mutations of the beta subunit of the cyclic guanosine monophosphate (cGMP) phosphodiesterase (beta-PDE) gene in rod photoreceptors of mice and dogs are similar to one of the inherited retinal degenerations termed retinitis pigmentosa in humans. Defects in the rod beta-PDE gene leading to photoreceptor cell degeneration in retinal degenerative (rd) mice can be corrected by transfer of a wild type beta-PDE gene. However, the rapid photoreceptor degeneration in this mutant makes the study of gene therapy difficult. Since the retinal degeneration is slowed in vitro, we have employed retinal explants from rd mice to study factors influencing viral transduction. Retinal explants provide a rapid, efficient method to compare the transduction efficiency of adenoviral vector-mediated reporter gene delivery at different ages in normal and rd mice. Retinal explants from postnatal day (P)2 to P28 control (C57BL/6J) and P2-P42 rd mice were exposed for 20 hr to 2.5 x 10(8) plaque forming units (pfu) ml(-1) of adenoviral vector with a beta-galactosidase (Lac Z) reporter gene (Ad-CMV-Lac Z). After incubation in vector-free media for an additional 3 days, the explants were fixed and histochemically stained for beta-galactosidase to reveal Lac Z gene expression. The explants were also embedded and sectioned for light microscopic observation. Transduction efficiency was higher in rd mice than in controls on all postnatal days examined. In normal retinal explants, expression of the Lac Z gene increased from P2 to a peak around P7-P8, then decreased at subsequent ages; little transduction could be found after P17. In rd mice transduction efficiency of Ad-CMV-Lac Z increased from P2 to P7, decreased by P10 and increased again after P10. The most dramatic increase in the transduction efficiency occurred in the rd retina between P10 and P15 when Lac Z was intensely expressed throughout the retina. Microscopic examination of retinal sections revealed the types and distribution of Lac Z-positive cells responsible for the deep blue staining in the retinal whole mount. In normal and rd mice, Lac Z-positive cells were located throughout the retina. However, larger numbers of Lac Z-positive cells were present at all ages examined in retinal explants from rd mice compared to normal mice. These data indicate a difference in transduction efficiency between normal and rd mice, especially after P12, and suggest efficient adenovirus-mediated gene transfer is more attainable in developing or degenerating retina. Thus, transduction efficiency in rd mice depends on the relationship between development, maturation and the degenerative state of the photoreceptor cells.     

Deletion of prominin-1 in mice results in disrupted photoreceptor outer segment protein homeostasis

AIM: To illustrate the underlying mechanism how prominin-1 (also known as Prom1) mutation contribute to progressive photoreceptor degeneration. METHODS: A CRISPR-mediated Prom1 knockout (Prom1-KO) mice model in the C57BL/6 was generated and the photoreceptor degeneration phenotypes by means of structural and functional tests were demonstrated. Immunohistochemistry and immunoblot analysis were performed to reveal the localization and quantity of related outer segment (OS) proteins. RESULTS: The Prom1-KO mice developed the photoreceptor degeneration phenotype including the decreased outer nuclear layer (ONL) thickness and compromised electroretinogram amplitude. Immunohistochemistry analysis revealed impaired trafficking of photoreceptor OS proteins. Immunoblot data demonstrated decreased photoreceptor OS proteins. CONCLUSION: Prom1 deprivation causes progressive photoreceptor degeneration. Prom1 is essential for maintaining normal trafficking and normal quantity of photoreceptor OS proteins. The new light is shed on the pathogenic mechanism underlying photoreceptor degeneration caused by Prom1 mutation.     

The retina of the PCD/PCD mouse as a model of photoreceptor degeneration. A structural and functional study

In this work, we used the pcd (Purkinje cell degeneration) mutant mouse with a slow temporal progression of photoreceptor degeneration in order to analyze the structural and functional modifications in the neuronal populations of the outer and inner retina.Retinal immunocytochemistry and functional electroretinography were performed on the pcd/pcd mutant mice and control wild type animals of the C57/DBA strain at 45, 90, 180 and 270 post-natal days. Immunohistochemical studies were performed for a series of protein markers: calbindin, calretinin, PKCα, bassoon, synapsin, syntaxin and islet1. Full field electroretinography recordings were performed on control and dystrophic mice. Rod and mixed responses, and oscillatory potentials, were recorded in dark adapted conditions; cone and flicker responses were recorded under light adaptation. Our results show significant structural modifications in the photoreceptor populations and neurons of the inner retina. Changes in cell morphology affect mainly to the bipolar cells, which gradually lose their dendritic tufts. The electroretinography records reveal that in the pcd retinas the rod and cone systems show a reduction in the amplitude of the electrical signals. This decrease progresses slowly with the passage of time, although for the most advanced stage of photoreceptor degeneration considered, 270 post-natal days, it is still possible to record light induced responses. We conclude that pcd mice experience a loss of retinal function in correlation with the loss of photoreceptors with age, and significant changes in retinal synaptic processes.     

Remodeling of cone photoreceptor cells after rod degeneration in rd mice

We studied the survival of cone photoreceptors following the degeneration of rods in the rd mouse. Cones were visualized by selective expression of green fluorescent protein (GFP) following transduction with an adeno-associated virus (AAV) vector. As previously reported, many cones survive after the initial degeneration of the rods. Soon after the initial degeneration, they lose their outer segments and all but a vestigial inner segment; and they partially retract or lose their axon and synaptic pedicle. However, they retain many fundamental features of the cone phenotype, and for many weeks show a polarized morphology indicative of substantial regrowth of processes. The cells retain their laminar position, forming a cell row just distal to a much thinned outer plexiform layer. The somata subsequently enlarge. Most of the cells extend bipolar processes, recreating the original bipolar morphology of a photoreceptor cell - though now turned on its side relative to the native position. The cells express short- or middle-wavelength opsins, recoverin and connexin36. One or more of the polarized processes could often be shown to contain synaptic ribbons, as visualized by antibodies against RIBEYE. The cones do not express protein kinase C alpha, Go alpha, ChX10 or calbindin, markers of bipolar or horizontal cells. The partially differentiated cone morphology persists for at least several months, after which the processes begin to retract and there is slow loss of the cells. Thus, during the time following the loss of their rod-dominated microenvironment, the cones achieve a semi-stable state in which much of their normal phenotype is preserved. Cone photoreceptors in retinas of human RP donors appear from their morphology to undergo a similar progression. The therapeutic window for rescue of cone photoreceptors may be longer than would have been thought.     

The zinc-binding domain of Nna1 is required to prevent retinal photoreceptor loss and cerebellar ataxia in Purkinje cell degeneration (pcd) mice

The Purkinje cell degeneration (pcd) mouse undergoes retinal photoreceptor degeneration and Purkinje cell loss. Nna1 is postulated to be the causal gene for pcd. We show that a BAC containing the Nna1 gene rescues retinal photoreceptor loss and Purkinje cell degeneration, confirming that Nna1 loss-of-function is responsible for these phenotypes. Mutation of the zinc-binding domain within the transgene destroyed its ability to rescue neuronal loss in pcd(5J) homozygous mice. In conclusion, Nna1 is required for survival of retinal photoreceptors and other neuron populations that degenerate in pcd mice. A functional zinc-binding domain is crucial for Nna1 to support neuron survival.     

快速退变性视网膜变性感光细胞超微结构变化分析

目的 了解快速退变性遗传性视网膜变性的感光细胞在出生后早期发生的形态学变化,为临床治疗提供依据.方法 取出生后不同时间的rd小鼠及正常对照小鼠各6只的视网膜,经光镜、扫描电镜和透射电镜观察感光细胞的形态发生发育和结构变化过程,比较二者的动态变化和形态学差异.结果 rd鼠生后1周开始出现感光细胞节段变短,内节段内线粒体变性改变多见;偶见感光细胞核旁胞浆内出现肿胀变形的线粒体.2周时节段层变薄,内节段结构已不完整,外节膜盘少见,变形且排列不整齐;外核层细胞层数明显减少,可见核固缩及染色质凝聚,偶见外丛状层部分神经突起内出现变性的线粒体.3周时节段层近消失,外核层只剩一层细胞,胞浆内可见髓样结构;外丛状层变薄.4周时内节段高度变形,视网膜色素上皮层与外核层之间出现大量不成形结构.外核层仅残存少许胞体,胞浆内出现多量不成形结构.外丛状层极薄,部分区域已消失.结论 rd鼠在出生后早期就发生感光细胞的变性改变,细胞内线粒体改变明显.其感光细胞变性发生早,进展快,呈快速退变特点.