遗传性视网膜变性rd小鼠及其感光细胞凋亡研究

目的 研究遗传性视网膜变性rd小鼠感光细胞层的发育变化及细胞凋亡。方法 对出生后5d到40d的rd小鼠及对照小鼠视网膜感光细胞层进行光镜及超微结构观察、TUNEL法检测及形态计量学分析。结果 与同龄对照鼠相比,rd小鼠出生后第10d视网膜开始变性,尔后1周内感光细胞迅速减少,第18d时只残留一层视椎细胞。rd小鼠出生后第10d感光细胞层开始出现TUNEL染色阳性细胞,第14d及16d达到高峰。电镜下变性高峰期rd小鼠视网膜感光细胞层可见大量浓缩核、染色质边聚及凋亡小体。结论 rd小鼠视网膜感光细胞在发育过程中变性,并通过凋亡的方式死亡。     

人视网膜小胶质细胞形态及分布的研究

本文用白细胞共同抗原（CD45）和巨噬细胞特异性抗原（CD68）的单克隆抗体，对24，28，30，33，36周胎龄及正常成人的视网膜进行标记。结果表明幼稚的阿米巴型，成熟的分支型和介于两者之间的中间型（依突起数可分为单极，双极，多极形的小胶质细胞）小胶质细胞均表达CD45，除分支型外，其余的小胶质细胞亦同时表达CD68，小胶质细胞分布于视网膜的内核层，内网层，节细胞层。在其分布的层内，细胞分布均匀，相邻的细胞之间无接触，节细胞层的细胞数量比内核层，内网层多，但内核层，内网层的细胞比节细胞层的细胞幼稚，形态上表现为胞体较大，细胞无分支或分支较少，无次级突起，同时发现随胎龄的增大，细胞数量增多，突起增多并变长变细，36周胎时已分化为分支型小胶质细胞，亦观察到血管周小胶质细胞和血管旁小胶质细胞，在成体视网膜小胶质细胞以分支型为主，同时也存在一系列形态较幼稚的CD68阳性细胞。     

RCS大鼠感光细胞凋亡与 Fas蛋白表达

为了探讨遗传性视网膜变性时感光细胞凋亡及其基因调控机制 ,本研究对出生后 9、15、2 0、2 5、3 0、3 5、40、60 d的 RCS大鼠及同龄 SD大鼠各 4只的视网膜进行了 TU NEL 凋亡检测及 Fas蛋白免疫组织化学反应。结果表明 ,出生后 2 5～ 40 d,RCS大鼠视网膜外核层可见 TUNEL阳性的感光细胞核 ,TUNEL阳性细胞数到 3 5 d达高峰 ( P<0 .0 5 )。Fas蛋白免疫组织化学检测发现 ,RCS大鼠视网膜内核层在 15～ 40 d可见 Fas免疫阳性细胞 ,阳性细胞数以 2 5 d为最多 ( P<0 .0 5 ) ;外核层在 2 5 d也可见Fas蛋白免疫阳性反应 ,一直持续到 40 d;节细胞层在 15～ 40 d可见 Fas蛋白表达。到 60 d时则各层又都不见明显的 Fas蛋白阳性反应。本研究结果提示 ,在 RCS大鼠视网膜变性过程中 ,感光细胞发生凋亡 ,Fas蛋白高表达可能与感光细胞的凋亡有关     

大鼠视网膜表达生长抑制mRNA细胞的发生

目的：研究视网膜中表达生长抑素mRNA神经元的发生和发育规律。方法：原位杂交组织化学。结果：胚胎第十三天（E13），视网膜内层就可以检测到生长抑素mRNA阳性细胞。从E14到E17阳性细胞增加迅速，E17达到高峰，此时视网膜节细胞层大部分为生长抑素mRNA阳性细胞，细胞排列紧密，从E18开始，阳性细胞逐渐减少，细胞排列开始稀疏。出生后当（PND0）阳性细胞数显著下降，从PND0到PND15，阳性细胞继续减少，阳性细胞主要位于节细胞层，PND20时，阳性细胞的数目及分布均与成年动物相似，部分已迁移至内核层的内表面。细胞开始或停止表达生长抑素mRNA均是从视网膜中央开始，然后向周围。结论：首次发现生长抑素基因在胚胎期的节细胞中一过性表达，提示生长抑素在视网膜节细胞的产生，分化以及视觉通路的形成与成熟有关。     

Caspase-9抑制剂对RCS大鼠感光细胞凋亡抑制作用的研究

目的：采用Z-LEHD-FMK进行体内实验，观察caspase-9抑制剂对RCS大鼠感光细胞凋亡的抑制作用。方法：32只 18 d RCS大鼠随机分4组，检查ERG后随机选择一眼为实验眼给予玻璃体内注射Z-LEHD-FMK 4 μg，对侧眼给予4 μg 2%DMSO作对照。各组分别在术后2 d、7 d、12 d、17 d行ERG检查，然后摘取双眼球，石蜡切片行HE染色及感光细胞凋亡的TUNEL检测，透射电镜观察视网膜超微结构。结果：实验眼注药后 7 d ERG b波振幅达到最大(137.35±7.41)mV，17 d时b波振幅为(57.91±9.27)mV，对照眼7 d及以后各组ERG接近熄灭型；实验眼注药后12 d视网膜外颗粒层才开始出现凋亡阳性细胞，17 d更明显，对照眼强荧光的凋亡阳性细胞在术后7 d已经很明显；光镜下注药后17 d实验眼感光细胞外颗粒层细胞数尚保持有7-8层，对照眼仅余下2-4层细胞，视网膜厚度变薄；透射电镜下实验眼注药后17 d可见部分感光细胞胞核、核仁固缩，对照眼从术后 7 d 开始见感光细胞呈现凋亡改变。结论：Z-LEHD-FMK能够延缓RCS大鼠感光细胞凋亡，合适的caspases抑制剂在适宜的时机应用对视网膜感光细胞凋亡具有一定的抑制作用。     

兔胎视网膜内P物质免疫反应神经元的发生

本文用免疫细胞化学ＡＢＣ法，研究了新西兰白兔１８、２２、２５、２６、２８和３０ｄ胎龄视网膜内Ｐ物质免疫反应（ＳＰＩＲ）神经元的发生。在胎龄１８和２２ｄ兔视网膜未见ＳＰＩＲ细胞体和纤维。在胎龄２５ｄ视网膜的节细胞层最先出现ＳＰＩＲ神经元，胞体浅染呈卵圆形，突起不明显，在神经纤维层偶见串珠状ＳＰＩＲ纤维，其平均细胞密度为１０４．６个细胞／ｍｍ￣２。到胎龄２６和２８ｄ时，在节细胞层的ＳＰＩＲ神经元的胞体渐深染，可见个别ＳＰＩＲ神经元发出粗而短的突起伸向内网层，平均细胞密度分别为３８７和７７９．５个细胞／ｍｍ２。到胎龄３０ｄ时ＳＰＩＲ神经元开始出现于内核层的内排细胞，但数量很少，胞体呈卵圆形，发出细突起伸入内同层，在节细胞层的ＳＰＩＲ神经元的突起分支增加。此时ＳＰＩＲ神经元平均细胞密度为３５７．４个细胞／ｍｍ￣２。     

生后小鼠不同年龄阶段视网膜片层化及胶质细胞发育特点

目的:观察小鼠视网膜片层化过程中胶质细胞增殖及与双极细胞分化的关系。方法:各个年龄小鼠共计123只。应用免疫荧光和HE染色法对各个年龄的小鼠视网膜形态结构及胶质细胞的增殖、分化进行观察。结果:星形胶质细胞按照离心型的模式发育,P0时仅在视乳头和邻近周围区域出现幼稚的星形胶质细胞,尔后细胞突起相互缠绕形成的网状联系中出现中空"管样"结构,细胞形态呈现典型的星状外形,类似血管网的脉络。P6时小鼠视网膜Müller细胞零星的表达GS,双极细胞也开始表达少量的PKC-α,尔后Müller细胞GS阳性表达逐渐增加,而PKC-α阳性细胞伸向节细胞层的轴突更加密集,树突和胞体的数量也增多,P14时双极细胞的发育过程基本完成。同时,小鼠视网膜GS和PKC-α双标记,发现Müller细胞内、外突上发出许多细小的侧突与双极细胞的胞体相接触,提示Müller细胞在双极细胞的分化、迁移过程中伴着很重要的角色。结论:在小鼠视网膜片层化过程中,胶质细胞起着关键性作用,Müller细胞在双极细胞的分化、迁移过程中起重要作用。     

兔视网膜中P物质样免疫反应神经元的发育

本实验用免疫细胞化学ABC法,研究了成年、新生和生后兔视网膜中P物质(SP)样免疫反应神经元的定位和发育。结果表明,成年兔视网膜SP样免疫反应细胞胞体位于内核层和节细胞层,胞突分布在内网层的第1、3、5亚层,偶见于视神经纤维层。细胞密度以视纹最高,从视纹向背腹视网膜边缘区密度渐变小。在新生兔视网膜已有SP阳性胞体和胞突出现,胞体主要位于节细胞层,突起在内网层第5亚层,但未形成连续网层,在第1亚层很少,第3亚层未见SP阳性突起。SP阳性细胞密度从新生到生后第4天增加,生后第6天到第12天细胞密度渐下降。生后第12天SP阳性胞体主要位于内核层。生后第20天,SP阳性细胞的形态、密度与分布已接近成年水平。上述结果提示,在兔视网膜中SP样免疫反应胞体和突起在生前已出现,生后继续发育,到生后20天后其形态发育已接近成熟。     

大鼠视网膜表达生长抑素mRNA细胞的发生

目的 :研究视网膜中表达生长抑素mRNA神经元的发生和发育规律。方法 :原位杂交组织化学。结果 :胚胎第十三天 (E1 3 ) ,视网膜内层就可以检测到生长抑素mRNA阳性细胞。从E1 4到E1 7阳性细胞增加迅速 ,E1 7达到高峰 ,此时视网膜节细胞层大部分为生长抑素mRNA阳性细胞 ,细胞排列紧密。从E1 8开始 ,阳性细胞逐渐减少 ,细胞排列开始稀疏。出生后当 (PND0 )阳性细胞数显著下降。从PND0到PND1 5 ,阳性细胞继续减少 ,阳性细胞主要位于节细胞层。PND2 0时 ,阳性细胞的数目及分布均与成年动物相似 ,部分已迁移至内核层的内表面。细胞开始或停止表达生长抑素mRNA均是从视网膜中央开始 ,然后向周围。结论 :首次发现生长抑素基因在胚胎期的节细胞中一过性表达 ,提示生长抑素在视网膜节细胞的产生 ,分化以及视觉通路的形成与成熟有关。     

视网膜祖细胞干细胞特性及移植入视网膜后的研究

目的：研究视网膜祖细胞的干细胞特性及移植入视网膜后的存活和迁移。方法：体外培养胎龄18d 大鼠的视网膜细胞,用 RT-PCR、细胞免疫荧光方法鉴定其增殖分化;成年 SD 大鼠腹腔注射 N-甲基-N-亚硝基脲形成视网膜感光细胞退化的动物模型,培养的视网膜祖细胞用 CM-Di(?) 标记后,移植入模型鼠的玻璃体腔。结果：视网膜祖细胞体外表达中间神经丝蛋白 nestin;表达 Flk-1、Pax6及 Notchl 的 mRNA;能掺入 BrdU;分化后表达视网膜各类细胞特异性蛋白;移植后在实验组大鼠视网膜能存活及迁移,而在对照组中仅聚集在玻璃体腔。结论：视网膜祖细胞具有干细胞特性,移植入受损伤视网膜后,能存活、整合及迁移。     

Morphological properties of mouse retinal ganglion cells

The mouse retina offers an increasingly valuable model for vision research given the possibilities for genetic manipulation. Here we assess how the structural properties of mouse retinal ganglion cells relate to the stratification pattern of the dendrites of these neurons within the inner plexiform layer. For this purpose, we used 14 morphological measures to classify mouse retinal ganglion cells parametrically into different clusters. Retinal ganglion cells were labeled in one of three ways: Lucifer Yellow injection, 'DiOlistics' or transgenic expression of yellow fluorescent protein. The resulting analysis of 182 cells revealed 10 clusters of monostratified cells, with dendrites confined to either On or Off sublaminae of the inner plexiform layer, and four clusters of bistratified cells, dendrites spanning the On and Off sublaminae. We also sought to establish how these parametrically identified retinal ganglion cell clusters relate to cell types identified previously on the basis of immunocytochemical staining and the expression of yellow fluorescent protein. Cells labeled with an antibody against melanopsin were found to be located within a single cluster, while those labeled with the SMI-32 antibody were in four different clusters. Yellow fluorescent protein expressing cells were distributed within 13 of the 14 clusters identified here, which demonstrates that yellow fluorescent protein expression is a useful method for labeling virtually the entire population of mouse retinal ganglion cells. Collectively, these findings provide a valuable baseline for future studies dealing with the effects of genetic mutations on the morphological development of these neurons.     

银杏苦内酯B对N-甲基-N-亚硝脲诱导的大鼠视网膜细胞凋亡的抑制作用

目的: 探讨银杏苦内酯B(GB)对N-甲基-N-亚硝脲(MNU)诱导的大鼠视网膜变性的影响及其机制。方法: 建立大鼠视网膜变性模型,应用TUNEL法检测光感受器细胞凋亡,RT-PCR法和免疫组织化学法分别检测MNU作用后不同时间大鼠视网膜中bcl-2和bax mRNA和蛋白的表达。结果: GB治疗组外核层细胞凋亡指数显著低于模型组(P<0.01)。MNU作用后12 h、1 d、2 d、3 d和5 d,bcl-2/bax mRNA模型组为0.36、0.15、0.29、0.42和0.64,GB治疗组为0.98、0.92、0.53、0.45和0.68,显著高于模型组(P<0.01)。GB治疗组Bcl-2蛋白在MNU给药后1 d表达最强,2 d阳性表达下降,3 d后阳性表达消失,模型组未见视网膜Bcl-2阳性表达;GB治疗组Bax蛋白表达显著低于模型组(P<0.01)。结论: 银杏苦内酯B能抑制MNU诱导的视网膜光感受器细胞凋亡,可能与上调Bcl-2的表达量,提高Bcl-2/Bax比值有关。     

黑视素基因转染给光双极细胞部分恢复MNU诱导的视网膜感光细胞变性小鼠的视觉

 目的： 探索定向转染内源性光感受蛋白黑视素（melanopsin/opsin 4,Opn4）基因进入给光型双极细胞后,视网膜变性小鼠模型中视网膜神经元的光反应以及整体视觉行为的改变。方法：选用由甲基亚硝基脲（N-methyl-N-nitrosourea,MNU）诱导的成年CD1小鼠作为视网膜变性模型。于P0~P1 CD1乳鼠视网膜底注射Grm6-Opn4-GFP质粒,Grm6-GFP作为阴性对照。通过电转进行基因转染。术后5周对基因转染小鼠腹腔注射MNU诱导视网膜感光细胞变性,对照组注射生理盐水,共设计5个实验组：正常对照组（normal）、生理盐水Grm6-Opn4-GFP对照组（NS+melanopsin）、MNU诱导模型Grm6-Opn4-GFP治疗组（MNU+melanopsin）、MNU诱导模型Grm6-GFP对照组（MNU+GFP）和MNU诱导组（MNU）。诱导后连续7 d进行明暗箱测试,统计动物在暗箱中的活动时间比。随后进行视网膜电图（electroretinogram,ERG）测试,计算体现给光双极细胞光反应的b波峰值、潜伏期和反映视网膜神经节细胞（retinal ganglion cells,RGCs）光反应的明视负波反应（photopic negative response,PhNR）。利用免疫荧光法检测动物视网膜黑视素基因转导效果。结果：黑视素可以被定向转染进入视网膜给光双极细胞。明暗箱实验显示MNU诱导7 d后Grm6-Opn4-GFP转染的CD1小鼠滞留在黑箱的时间显著长于未转染组（P<0.05）,ERG测试显示Grm6-Opn4-GFP转染的CD1小鼠的b波也有明显恢复（P<0.05）。结论：定向转染内源性光感受蛋白黑视素基因进入给光型双极细胞可部分恢复视网膜变性模型动物视觉。     

Expression of the neurokinin 1 receptor in the rabbit retina

Substance P is the preferred ligand for the neurokinin 1 (NK1) receptor. In vertebrate retinas, substance P is expressed by amacrine, interplexiform and ganglion cells. Substance P influences the activity of amacrine and ganglion cells and it is reported to evoke dopamine release. We investigated NK1 receptor expression in the rabbit retina using affinity-purified NK1 receptor antibodies. NK1 receptors were expressed by two distinct populations of retinal neurons. One is a population of ON-type bipolar cells characterized by axonal arborizations that ramified in the inner plexiform layer near the ganglion cell layer. Double-label studies showed that NK1 receptor-expressing bipolar cells were distinct from rod bipolar cells and from other immunocytochemically identified types of cone bipolar cells. Their density was about 2250 cells/mm2 in the visual streak and 1115 cells/mm2 in ventral mid-periphery. They were distributed in a non-random pattern. In the outer plexiform layer, the dendrites of these bipolar cells converged into heavily immunostained clusters having a punctate appearance. The density of these clusters in mid-peripheral ventral regions (about 13000 clusters/mm2) was similar to the reported cone density [Famiglietti and Sharpe (1995) Vis. Neurosci. 12, 1151-1175], suggesting these dendrites contact all cone photoreceptors. The second NK1 receptor expressing cell population corresponds to the tyrosine hydroxylase-containing amacrine cell population. NK1 receptor immunostaining was localized to the cell body and processes, but not to the processes that form the 'rings' that are known to encircle somata of AII amacrine cells. These findings show that NK1 receptor immunoreactivity is localized to a population of ON-type cone bipolar cells and to dopaminergic amacrine cells, suggesting that substance P acting on NK1 receptors influences multiple retinal circuits in the rabbit retina.     

Phototransduction in ganglion-cell photoreceptors

A third class of photoreceptors has recently been identified in the mammalian retina. They are a rare cell type within the class of ganglion cells, which are the output cells of the retina. These intrinsically photosensitive retinal ganglion cells support a variety of physiological responses to daylight, including synchronization of circadian rhythms, modulation of melatonin release, and regulation of pupil size. The goal of this review is to summarize what is currently known concerning the cellular and biochemical basis of phototransduction in these cells. I summarize the overwhelming evidence that melanopsin serves as the photopigment in these cells and review the emerging evidence that the downstream signaling cascade, including the light-gated channel, might resemble those found in rhabdomeric invertebrate photoreceptors.     

Synapsin I expression in the rat retina during postnatal development

Summary The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.Abbreviations GCL ganglion cell layer - INB inner neuroblast layer - INL inner nuclear layer - IPL inner plexiform layer - ONB outer neuroblast layer - ONL outer nuclear layer - OPL outer plexiform layer     

细菌脂多糖诱导小鼠DC2.4细胞TLR7蛋白的表达

目的:探讨细菌脂多糖(LPS)对小鼠树突状细胞(DC)系DC2.4 TLR7蛋白表达的诱导作用.方法:用RPMIl640完全培养液培养DC2.4细胞,光学显微镜观察LPS刺激前后的细胞形态特征,RT-PCR和Western blot分别测定TLR7 mRNA和蛋白表达.结果:LPS刺激前后,细胞中均有TLR7 mRNA转录.LPS刺激前,细胞较小而透亮,树突较少,无,TLR7蛋白表达;LPS刺激后,细胞体积增大,树突增粗增多,刺激后12 h、24 h、48 h,TLR7蛋白均有表达,且在3个时间点的表达量基本一致.结论:LPS可诱导DC2.4中TLR7蛋白的表达.