Basic research for clinical application of retina and optic nerve regeneration by transplantation

视网膜及视神经移植再生临床应用基础研究

• 批准号: 07307016
• 负责人: TAMAI Makoto
• 金额: $ 12.99万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07307016/
• 关键词: regeneration; transplantation; optic nerve; sciatic nerve; retina; retinal pigement epithelium; Muller cells; neurotrophic factors

1) Degeneration process of retinal ganglion cells : By means of confocal laser scanning microscope, both dendritic and axonal processes located in different focal planes can be observed at a single image without any blur. Without sectioning flat-mounted retinas vertically, confocal laser scanning microscope enabled us to identify either on-type cells or off-type cells clearly and easily on the basis of their reconstructed vertical image.2) Axonal regeneration of retinal ganglion cells : We concluded that even adult aged retinal ganglion cells had the ability of axonal regeneration after injury and that neurotrophic factors might enhanced these abilities. Therefore neurotrophic factors might have practicable appiocations in drug treatments for intractable disease of the neural retina and optic nerve.3) Observation of possible cases for transplantation : Using a scanning laser ophthalmoscope (SLO) , the ocular fundus of a patient with Best's vitelliform maculardystrophywas examined. With … More the argon blue laser, a dark area was seen in the fovea. Areas of weakly bright reflex were seen corresponding to the scrambled-egg lesion observed by ophthalmoscope. With the argon green laser, highly bright spots were observed at the foveola. Intense bright reflex was seen over the scrambled-egg lesion at theposterior pole. The helium-neon laser revealed highly bright patches corresponding to the orderly ring distribution of the yellowish deposits at the posterior pole. The diode laser revealed a mottled appearance with dark and bright patches in the scrambled-egg lesion. SLO provided a morphologic enhancement in the specifically affected layrs of the maculs in Best's vitelliform macular dystrophy.4) Analysis of retinal regeneration using avian embryo : The avian pineal is a photoreceptive organ and is believed to function as a circadian clock. Avian pineal cells are secretory rudimentary photoreceptors, and previous studies have demonstrated that there are two types of photoreceptors in developing quail pineals, one of which is rhodopsin-like immunoreactive and the other iodopsin-like immunoreactive. Much larger number of rhodopsin-like immunoreactive cells than of iodopsin-like immunoreactive cells were found in quail pineals, both in vivo and in vitro. In the present study we co-cultured pineal cells of embryonic quails with retinal cells but separated the two with a biomembrane filter We found that diffusible material produced by the cultured retinal cells intensely promotes the appearance of pineal iodopsin-like immunoreactive cells in vitro. This effect of retina-derived factors) is cell-type specific, since there is no effect on the differentiation of pineal rhodopsin-like immunoreactive cells. Retinal cell cultures had much more intensive iodopsin-promoting effect than other embryonic brain cultures such as cerebral cell cultures. The production of the retinal factors) seems to be developmentally regulated, since retiual cells from older embryos (E13 and older) did not have such effects. The factors) possibly act on pineal precursor cells by stimulating the expression of the iodopsin-like immunoreactive phenotype. Preliminary characterization of conditioned medium obtained from cultured retinal cells shows that the factor is a stable polypeptide, probably of low molecular weight. The pineal-retina culture system win provide a good experimental system to analyze the effect of extrinsic environments on cell differentiation.5) Physiology of the retinal regeneration : Retinal cells dissociated from adult newt eyes were seeded onto plastic dishes which were coated with various cell adhesion substances. They were cultured in Leibovitz's L-15 medium with no serum. Concanavalin A (ConA) and poly-L-lysine (PLL) caused relatively good cell attachment, regardless of retinal cell types, in comparison with uncoated dishes. On the other hand, extracellular matrix (ECM) components such as collagen, fibronectin and laminin tended to inhibit the cell attachment. In both ConA-and PLL-coated dishes, ganglion cells survived for longer periods than other retinal cells and extended neurites with time in culture. The outgrowth of neurites by ganglion cells was found to be promoted to a far greater degree by ConA than PLL.Branching of neurites also appeared to occur more frequently on ConA.From these observations, ConA may be an useful tool for investigating retinal regeneration in vitro. Less

1)视网膜神经节细胞的变性过程：利用共聚焦激光扫描显微镜，可以在同一图像上观察到位于不同焦平面的树突和轴突过程，没有任何模糊。共聚焦激光扫描显微镜不需要对视网膜进行垂直切片，就能根据重建的垂直图像清晰、方便地识别出on-type细胞和off-type细胞。2）视网膜神经节细胞的轴突再生：我们认为成年老年视网膜神经节细胞在损伤后也具有轴突再生的能力，神经营养因子可能增强这种能力。因此，神经营养因子可能在神经视网膜和视神经的难治性疾病的药物治疗中具有可行的应用。3）可能的移植病例的观察：用扫描激光检眼镜（SLO）对1例Best卵黄样黄斑营养不良患者的眼底进行了检查。与 ...更多信息 氩蓝激光，在中央凹中看到一个暗区。眼底镜下可见与搅蛋样病变相对应的弱亮反射区。使用氩绿色激光，在小凹处观察到高度明亮的斑点。在后极的搅蛋状病变上可见强烈的明亮反射。氦氖激光显示出非常明亮的斑块，对应于后极黄色沉积物的有序环形分布。半导体激光显示杂乱的鸡蛋病变中有暗斑和亮斑的斑驳外观。SLO在Best卵黄状黄斑营养不良中的黄斑的特定受影响层中提供形态学增强。4）使用禽类胚胎的视网膜再生的分析：禽类松果体是光感受器官并且被认为起生物钟的作用。鸟类松果体细胞是一种分泌型的初级光感受器，以往的研究表明，在发育中的鹌鹑松果体中有两种类型的光感受器，其中一种是视紫红质样免疫反应性的，另一种是碘视蛋白样免疫反应性的。在鹌鹑松果体中，视紫红质样免疫反应细胞的数量明显多于视碘质样免疫反应细胞。本研究将鹌鹑胚胎松果体细胞与视网膜细胞共培养，用生物膜过滤器将两者分离，发现视网膜细胞产生的可扩散物质强烈促进松果体碘视素样免疫反应细胞的出现。视网膜衍生因子的这种作用是细胞类型特异性的，因为对松果体视紫红质样免疫反应细胞的分化没有影响。视网膜细胞培养物对碘视蛋白的促增殖作用明显强于其他胚胎脑细胞培养物。视网膜因子的产生似乎是受发育调节的，因为来自较老胚胎（E13和更老）的视网膜细胞没有这种作用。这些因子可能通过刺激视碘蛋白样免疫反应表型的表达而作用于松果体前体细胞。从培养的视网膜细胞获得的条件培养基的初步表征表明，该因子是一种稳定的多肽，可能是低分子量的。视网膜再生的生理学：将成年蝾螈视网膜细胞分离，接种于涂有不同细胞粘附物质的培养皿中，观察视网膜细胞的再生情况。将它们培养在不含血清的Leibovitz L-15培养基中。伴刀豆球蛋白A（ConA）和聚-L-赖氨酸（PLL）引起相对良好的细胞附着，无论视网膜细胞类型，与未涂层的盘子相比。另一方面，细胞外基质（ECM）成分，如胶原蛋白、纤连蛋白和层粘连蛋白往往会抑制细胞附着。在ConA和PLL包被的培养皿中，神经节细胞比其他视网膜细胞存活时间更长，并且随着培养时间的推移而延长神经突。ConA对神经节细胞突起的生长的促进作用远大于PLL，ConA也能更频繁地促进神经突起的分支。少

项目成果

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Yamada K、Mashima Y、Kigasawa K、Miyashita K、Wakakura M、Oguchi Y：“四名患有 14484 突变的莱伯遗传性视神经病的日本患者视力恢复率很高。”

Chiba, C., Sakai, H., Kaneko, Y.and Saito, T.: "(1995) Concanavaline A enhances neurite ougrowth of isolated ganglion cells from the adult newt retina." Zool.Sci.12. 741-747 (1995)

Chiba, C.、Sakai, H.、Kaneko, Y. 和 Saito, T.：“(1995) Concanavaline A 增强了成年蝾螈视网膜中分离的神经节细胞的神经突生长。”

Chiba, C.: "Time course of appearance of GABA and GABA-receptors during retinal regeneration in the adultnewt." Dev.Brain Res.98. 204-210 (1997)

Chiba, C.：“成虫视网膜再生过程中 GABA 和 GABA 受体出现的时间过程。”

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Kimura,T: "Molecular cloning and characterization of a putative neural calcium channel alpha1-subunit from squid optic lobe." Biochem.Biophysi.Res.Comm.230. 147-154 (1997)

Kimura,T：“鱿鱼视叶中假定的神经钙通道 α1 亚基的分子克隆和表征。”