两种自发性视网膜退行性变动物的形态学研究
发布时间：2019-06-08 10:31
【摘要】：视网膜遗传性疾病为危害严重且常见的眼科致盲性疾病，其中先天性静止性夜盲(congenital stationary night blindness,CSNB)以及视网膜色素变性(retinitis pigmentosa,RP)为其中较为常见的两种疾病。动物模型做为研究视网膜遗传性疾病的一种重要手段，其作用至关重要，我们实验室早期发现了关于这两种疾病的两个动物模型，即先天性静止性夜盲模式大鼠（简称CSNB大鼠）及快速视网膜变性（retinal degeneration fast，rdf）模式小鼠（简称rdf小鼠）。CSNB大鼠遗传方式为性连锁隐形遗传，其突变基因为Cacna1f基因； rdf小鼠为常染色体隐性遗传动物模型，突变基因为Pde6b基因。本实验主要观察CSNB模式大鼠出生后发育阶段其视网膜中间神经元之一水平细胞的变化，以及rdf模式小鼠出生后发育阶段其视神经髓鞘的变化，并探讨其可能的机制，旨在为遗传性视网膜疾病、视网膜视觉信号回路以及视觉神经信号传导的研究提供基本的实验依据。 方法 1.野生型SD大鼠及CSNB大鼠于出生后以随机化原则按将要处死的时间点出生后15天（postnatal day15， PND15）、出生后30天（PND30）、出生后60天（PND60）分组。 2.应用病理学染色方法、免疫组织化学染色方法、免疫组织荧光染色方法、全视网膜铺片免疫荧光染色方法及western blotting方法观察及对比野生型SD大鼠与CSNB大鼠出生后发育时期中其视网膜中水平细胞的变化。 3.野生型昆明种小鼠及rdf小鼠按照处理方式的不同（饲养于正常循环光照环境下及饲养于绝对暗室中）及将要处死的时间点（出生后7天、出生后14天、出生后28天，PND7、PND14、PND28）分组。按处理方式共分有4个组：野生型昆明小鼠对照组，rdf小鼠组，饲养于绝对暗室的野生型昆明小鼠对照组，饲养于绝对暗室的rdf小鼠组；其中各组内又按处死时间分为PND7，PND14，PND28这3个时间点。并建立绝对避光暗室，将暗室组小鼠于出生当天（PND0）即饲养于绝对暗室中。 4.应用病理学染色方法及电子显微镜方法观察野生型昆明种对照小鼠及rdf小鼠在出生后视觉发育时期中视神经直径、视神经髓鞘有髓纤维数目、直径及厚度的变化。 结果 1. CSNB大鼠的研究结果 CSNB大鼠在出生后15天、30天及60天（PND15,PND30，PND60）其视网膜中水平细胞数目及水平细胞在视网膜中的密度以及Calbindin蛋白的表达水平均低于同年龄组野生型SD大鼠（P 0.05）；野生型SD大鼠视网膜水平细胞数目及水平细胞的密度在各年龄时间点保持稳定，而CSNB大鼠视网膜水平细胞数目及水平细胞的密度均随着动物年龄的增长呈减少趋势；野生型SD大鼠视网膜中Calbindin蛋白的表达水平随着动物年龄的增长呈增加趋势，而CSNB大鼠视网膜中Calbindin蛋白的表达水平在各年龄时间点没有明显的差异；CSNB大鼠水平细胞轴突较野生型SD大鼠稀疏；CSNB大鼠视网膜中内核层与外丛状层之间检测到细胞凋亡；各组CSNB大鼠视网膜外丛状层厚度均低于同年龄组野生型SD大鼠（P 0.05）。 2.视网膜退行性变和光线对小鼠视神经髓鞘发育变化的影响结果 视神经直径：各组内视神经平均直径随年龄增长呈增加的趋势。在出生后7天，，14天和28天（PND7, PND14和PND28），野生型昆明小鼠以及饲养于暗室中的野生型昆明小鼠在各时间点没有明显差异。rdf小鼠其视神经直径明显小于同年龄组野生型昆明小鼠（P 0.05）。饲养于暗室中的rdf小鼠明显大于同年龄组rdf小鼠（P 0.05），但是和同年龄组饲养于暗室中的野生型昆明小鼠比较没有明显差异。 有髓髓鞘数目：野生型昆明小鼠与同年龄组饲养于暗室中的野生型昆明小鼠相比显著减少（P 0.05），rdf小鼠与同年龄组野生型昆明小鼠相比显著增加（P 0.05）。饲养于暗室中的rdf小鼠与同年龄组饲养于暗室中的野生型昆明小鼠相比显著减少（P 0.05）。 有髓髓鞘直径：在PND14及PND28，野生型昆明小鼠显著大于同年龄组饲养于暗室中的野生型昆明小鼠（P 0.05）。rdf组小鼠显著小于同年龄组野生型昆明小鼠（P 0.05）。饲养于暗室中的rdf组小鼠显著小于同年龄组rdf小鼠（P 0.05），而显著大于同年龄组饲养于暗室中的野生型昆明小鼠（P 0.05）。 有髓髓鞘厚度：(1)小轴突组，rdf小鼠显著大于同年龄组野生型昆明小鼠（P 0.05）。饲养于暗室中的野生型昆明小鼠显著大于同年龄组昆明小鼠（P 0.05）。(2)大轴突组中，rdf小鼠显著小于同年龄组野生型昆明小鼠（P 0.05）。饲养于暗室中的野生型昆明组小鼠显著小于同年龄组昆明小鼠（P 0.05）。(3)小直径组及大直径组中，均发现在PND14d时，饲养于暗室中的rdf小鼠显著小于同年龄组rdf小鼠（P 0.05），且显著小于饲养于暗室中的野生型昆明小鼠（P 0.05），但是在PND28d时饲养于暗室中的rdf小鼠显著大于同年龄组rdf小鼠（P 0.05），且显著大于饲养于暗室中的野生型昆明小鼠（P 0.05）。 结论 通过本研究的实验发现，CSNB大鼠中水平细胞的数目与野生型SD大鼠相比减少，并且其数目随着年龄的增长表现了下降的趋势，外丛状层厚度变薄，提示了先天性静止性夜盲可以引起视网膜水平细胞缺失并且抑制视网膜水平细胞的发育。 小鼠中视神经髓鞘的发育在一定程度上受到光剥夺和视网膜退行性变的独立负性影响，但是光剥夺对rdf小鼠的视神经髓鞘的抑制有一定的延缓作用，提示了光剥夺及视网膜退行性变各自都在一定程度上抑制了视神经髓鞘的发育，但是发育阶段的光剥夺却可以在一定程度上延缓视网膜变性疾病视神经髓鞘发育的改变。 本实验的研究结果提示，基因缺陷导致的遗传性疾病在视觉发育期即可影响视觉回路的信号传递，不仅仅局限于疾病基因表达的靶组织和细胞结构，由于视觉回路是一个复杂而精细的整体关联的结构，疾病也影响到视觉回路的其他部位，本实验的结果也进一步提示到，在视觉发育期，是对疾病发展以及视觉回路信号传递的过程进行干预和处理的关键时期，提示了在这一时期进行治疗时机可能更为关键并且治疗效果可能更为显著。
[Abstract]:The hereditary disease of the retina is a serious and common ocular blindness disease, in which the congenital static night blindness (CSNB) and the retinitis pigmentosa (RP) are two more common diseases. The animal model is an important means to study the hereditary diseases of the retina, and its function is of great importance. In the early stage of our lab, we found two animal models of the two kinds of diseases, namely, the congenital still night blindness mode (CSNB rats) and the rapid retinal degeneration. Rdf) model mice (rdf mice). The genetic pattern of CSNB rats is the inheritance of sex-linked genes. The mutant gene of CSNB is the Cacna1f gene, and the rdf mouse is the autosomal recessive genetic animal model, and the mutant gene is the Pde6b gene. This experiment mainly observed the changes of the level cells of one of the central retinal neurons of the rat at the postnatal development stage of the CSNB model, and the changes of the optic nerve pulp during the postnatal development stage of the rdf-mode mouse, and discussed the possible mechanism of the mechanism, aiming at providing the hereditary retinal disease, The visual signal loop of the retina and the conduction of the visual nerve signal provide a basic experimental basis. Method 1. Wild-type SD rats and CSNB rats were born 15 days after birth with the principle of randomization (postnatalday15, PND15),30 days after birth (PAND30), and 60 days after birth (PND60 ) Group2.2. Application of pathological staining method, immunohistochemical staining method, immune tissue fluorescence staining method, whole-retinal flap immunofluorescence staining method and western blotting method to observe and compare the levels of the wild-type SD rat and the CSNB rat in the post-natal development period 3. The changes of the cells.3. The wild-type Kunming mice and the rdf mice were different according to the treatment methods (housed in the normal circulating light environment and in the absolute dark room) and the time points to be sacrificed (7 days after birth,14 days after birth,28 days after birth, PND7, PND14, P, ND28) The group was divided into 4 groups according to the treatment method: wild-type Kunming mouse control group, rdf mouse group, wild-type Kunming mouse control group fed to the absolute dark room, rdf mouse group fed to the absolute dark room, and each group was divided into PAND7, PND14 and PND2 according to the sacrifice time. 8 The 3 time points were established and an absolute dark room was established, and the dark room group mice were fed on the day of birth (PND0). 4. The diameter of the optic nerve and the number of myelin in the optic nerve of the wild-type Kunming-type control mice and the rdf mice were observed in the visual development of the wild-type Kunming-type control mice and the rdf mice by means of a pathological staining method and an electron microscope method. , straight Changes in diameter and thickness. Results 1. The number of horizontal cells and the expression of Calbindin in the retina of CSNB rats were lower than that of the same age at 15,30 and 60 days after birth (PND15, PND30, PND60). Group wild-type SD rats (P 0.05); the number of retinal horizontal cells and the density of the horizontal cells in the wild-type SD rats remained stable at all age points, while the number of retinal horizontal cells and the density of the horizontal cells in the CSNB rats The expression level of Calbindin in the retina of the wild-type SD rats increased with the increase of the age of the animals, while the expression level of Calbindin in the retina of the CSNB rats was not significantly different at all ages; the water in the CSNB rats The axons of the flat cells were sparse in the wild type SD rats, and the apoptosis of the cells was detected between the inner core layer and the outer cluster layer in the retina of the CSNB rats, and the thickness of the outer plexiform layer of the CSNB rats in each group was lower than that of the same age. Group wild-type SD rats (P 0.05).2. Degenerative and light of the retina The effect of the line on the development of the optic nerve of the optic nerve of the mouse: the diameter of the optic nerve: each group The average diameter of the internal optic nerve increased with age. On 7,14 and 28 days after birth (PND7, PND14, and PND28), wild-type Kunming mice, and in the dark room There was no significant difference in the time points of wild-type Kunming mice. The diameter of the optic nerve in the rdf mice was significantly smaller. In the same age group, wild-type Kunming mice (P 0.05) were found in the same age group. The rdf mice fed in the dark room were significantly larger than that of the same age group rdf mice (P 0.05). However, the rdf mice in the same age group were fed in the same age group. There was no significant difference in the wild-type Kunming mice in the dark room. The number of myeloid cells: the wild-type Kunming mice significantly decreased compared with the wild-type Kunming mice fed in the dark room (P 0.05), rdf mice and the same age group. Compared with the wild-type Kunming mice (P 0.05), the rdf mice fed in the dark room and the same age group were housed in the dark room. Significant decrease in the wild-type Kunming mice (P 0.05). Myeloid diameter: in PND14 and PND28, wild-type Kunming mice were significantly larger than the same year The age group was housed in the wild-type Kunming mouse (P 0.05) in the dark room. The rdf group was small. The mice were significantly smaller than that of the wild-type Kunming mice in the same age group (P 0.05). The rdf group mice fed in the dark room were significantly smaller than the same age group rdf mice (P 0.05), which were significantly larger than that of the same age group (P 0.05). The age group was housed in the wild-type Kunming mouse (P 0.05) in the dark room. The thickness of the medullary canal: (1) the small axon group, rd F The mice were significantly larger than the wild-type Kunming mice in the same age group (P 0.05). Kunming mice of the same age group were significantly higher than those in the same age group (P 0.05). (2) in the large axon group, rd F The mice were significantly smaller than the wild-type Kunming mice in the same age group (P 0.05). The mice in Kunming group were significantly smaller than that of Kunming mice of the same age group (P 0.05). (3) In the small diameter group and the large diameter group, the rdf mice fed in the dark room were significantly smaller than the rdf mice in the same age group (P 0.05). The wild-type Kunming mice (P 0.05) were significantly smaller in the dark room than in the dark room, but the rdf mice fed in the dark room at the time of PND28d were significantly larger than the rdf mice in the same age group (P 0.05), and significant The results of this study show that the number of horizontal cells in CSNB rats is less than that of wild-type SD rats. And the thickness of the outer plexiform layer is thin, and the congenital static property is prompted. Night blindness can cause the loss of retinal horizontal cells and inhibit the development of retinal horizontal cells. The development of the optic nerve pulp in the mouse is affected by light deprivation and the independent negative of the degeneration of the retina. The effect of light deprivation on the optic nerve canal of the rdf mouse has a certain delay, and it is suggested that the optical deprivation and the degeneration of the retina have the effect of inhibiting the development of the optic nerve canal, but the development stage The results of this experiment suggest that the genetic disease caused by the gene defect can affect the signal transmission of the visual loop during the visual development period, not only to the disease gene table. The target tissue and cell structure of the invention, because the visual loop is a complex and delicate whole-related structure, the disease also affects the other parts of the visual loop, the results of the experiment also prompt to, in the visual development Period is a critical period for intervention and processing of the process of disease progression and the delivery of visual loop signals
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1
本文编号：2495215
[Abstract]:The hereditary disease of the retina is a serious and common ocular blindness disease, in which the congenital static night blindness (CSNB) and the retinitis pigmentosa (RP) are two more common diseases. The animal model is an important means to study the hereditary diseases of the retina, and its function is of great importance. In the early stage of our lab, we found two animal models of the two kinds of diseases, namely, the congenital still night blindness mode (CSNB rats) and the rapid retinal degeneration. Rdf) model mice (rdf mice). The genetic pattern of CSNB rats is the inheritance of sex-linked genes. The mutant gene of CSNB is the Cacna1f gene, and the rdf mouse is the autosomal recessive genetic animal model, and the mutant gene is the Pde6b gene. This experiment mainly observed the changes of the level cells of one of the central retinal neurons of the rat at the postnatal development stage of the CSNB model, and the changes of the optic nerve pulp during the postnatal development stage of the rdf-mode mouse, and discussed the possible mechanism of the mechanism, aiming at providing the hereditary retinal disease, The visual signal loop of the retina and the conduction of the visual nerve signal provide a basic experimental basis. Method 1. Wild-type SD rats and CSNB rats were born 15 days after birth with the principle of randomization (postnatalday15, PND15),30 days after birth (PAND30), and 60 days after birth (PND60 ) Group2.2. Application of pathological staining method, immunohistochemical staining method, immune tissue fluorescence staining method, whole-retinal flap immunofluorescence staining method and western blotting method to observe and compare the levels of the wild-type SD rat and the CSNB rat in the post-natal development period 3. The changes of the cells.3. The wild-type Kunming mice and the rdf mice were different according to the treatment methods (housed in the normal circulating light environment and in the absolute dark room) and the time points to be sacrificed (7 days after birth,14 days after birth,28 days after birth, PND7, PND14, P, ND28) The group was divided into 4 groups according to the treatment method: wild-type Kunming mouse control group, rdf mouse group, wild-type Kunming mouse control group fed to the absolute dark room, rdf mouse group fed to the absolute dark room, and each group was divided into PAND7, PND14 and PND2 according to the sacrifice time. 8 The 3 time points were established and an absolute dark room was established, and the dark room group mice were fed on the day of birth (PND0). 4. The diameter of the optic nerve and the number of myelin in the optic nerve of the wild-type Kunming-type control mice and the rdf mice were observed in the visual development of the wild-type Kunming-type control mice and the rdf mice by means of a pathological staining method and an electron microscope method. , straight Changes in diameter and thickness. Results 1. The number of horizontal cells and the expression of Calbindin in the retina of CSNB rats were lower than that of the same age at 15,30 and 60 days after birth (PND15, PND30, PND60). Group wild-type SD rats (P 0.05); the number of retinal horizontal cells and the density of the horizontal cells in the wild-type SD rats remained stable at all age points, while the number of retinal horizontal cells and the density of the horizontal cells in the CSNB rats The expression level of Calbindin in the retina of the wild-type SD rats increased with the increase of the age of the animals, while the expression level of Calbindin in the retina of the CSNB rats was not significantly different at all ages; the water in the CSNB rats The axons of the flat cells were sparse in the wild type SD rats, and the apoptosis of the cells was detected between the inner core layer and the outer cluster layer in the retina of the CSNB rats, and the thickness of the outer plexiform layer of the CSNB rats in each group was lower than that of the same age. Group wild-type SD rats (P 0.05).2. Degenerative and light of the retina The effect of the line on the development of the optic nerve of the optic nerve of the mouse: the diameter of the optic nerve: each group The average diameter of the internal optic nerve increased with age. On 7,14 and 28 days after birth (PND7, PND14, and PND28), wild-type Kunming mice, and in the dark room There was no significant difference in the time points of wild-type Kunming mice. The diameter of the optic nerve in the rdf mice was significantly smaller. In the same age group, wild-type Kunming mice (P 0.05) were found in the same age group. The rdf mice fed in the dark room were significantly larger than that of the same age group rdf mice (P 0.05). However, the rdf mice in the same age group were fed in the same age group. There was no significant difference in the wild-type Kunming mice in the dark room. The number of myeloid cells: the wild-type Kunming mice significantly decreased compared with the wild-type Kunming mice fed in the dark room (P 0.05), rdf mice and the same age group. Compared with the wild-type Kunming mice (P 0.05), the rdf mice fed in the dark room and the same age group were housed in the dark room. Significant decrease in the wild-type Kunming mice (P 0.05). Myeloid diameter: in PND14 and PND28, wild-type Kunming mice were significantly larger than the same year The age group was housed in the wild-type Kunming mouse (P 0.05) in the dark room. The rdf group was small. The mice were significantly smaller than that of the wild-type Kunming mice in the same age group (P 0.05). The rdf group mice fed in the dark room were significantly smaller than the same age group rdf mice (P 0.05), which were significantly larger than that of the same age group (P 0.05). The age group was housed in the wild-type Kunming mouse (P 0.05) in the dark room. The thickness of the medullary canal: (1) the small axon group, rd F The mice were significantly larger than the wild-type Kunming mice in the same age group (P 0.05). Kunming mice of the same age group were significantly higher than those in the same age group (P 0.05). (2) in the large axon group, rd F The mice were significantly smaller than the wild-type Kunming mice in the same age group (P 0.05). The mice in Kunming group were significantly smaller than that of Kunming mice of the same age group (P 0.05). (3) In the small diameter group and the large diameter group, the rdf mice fed in the dark room were significantly smaller than the rdf mice in the same age group (P 0.05). The wild-type Kunming mice (P 0.05) were significantly smaller in the dark room than in the dark room, but the rdf mice fed in the dark room at the time of PND28d were significantly larger than the rdf mice in the same age group (P 0.05), and significant The results of this study show that the number of horizontal cells in CSNB rats is less than that of wild-type SD rats. And the thickness of the outer plexiform layer is thin, and the congenital static property is prompted. Night blindness can cause the loss of retinal horizontal cells and inhibit the development of retinal horizontal cells. The development of the optic nerve pulp in the mouse is affected by light deprivation and the independent negative of the degeneration of the retina. The effect of light deprivation on the optic nerve canal of the rdf mouse has a certain delay, and it is suggested that the optical deprivation and the degeneration of the retina have the effect of inhibiting the development of the optic nerve canal, but the development stage The results of this experiment suggest that the genetic disease caused by the gene defect can affect the signal transmission of the visual loop during the visual development period, not only to the disease gene table. The target tissue and cell structure of the invention, because the visual loop is a complex and delicate whole-related structure, the disease also affects the other parts of the visual loop, the results of the experiment also prompt to, in the visual development Period is a critical period for intervention and processing of the process of disease progression and the delivery of visual loop signals
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1
【参考文献】
相关期刊论文 前3条
1 赵辉,尹志勇,王正国;视觉发育可塑性的电生理及分子生物学研究进展[J];国际眼科杂志;2004年06期
2 张馨方;盛迅伦;;视网膜色素变性的相关基因研究进展[J];国际眼科杂志;2006年03期
3 睢瑞芳;赵家良;;先天性静止性夜盲[J];中华眼科杂志;2006年05期
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