【摘要】：視網(wǎng)膜遺傳性疾病為危害嚴(yán)重且常見的眼科致盲性疾病，其中先天性靜止性夜盲(congenital stationary night blindness,CSNB)以及視網(wǎng)膜色素變性(retinitis pigmentosa,RP)為其中較為常見的兩種疾病。動(dòng)物模型做為研究視網(wǎng)膜遺傳性疾病的一種重要手段，其作用至關(guān)重要，我們實(shí)驗(yàn)室早期發(fā)現(xiàn)了關(guān)于這兩種疾病的兩個(gè)動(dòng)物模型，即先天性靜止性夜盲模式大鼠（簡稱CSNB大鼠）及快速視網(wǎng)膜變性（retinal degeneration fast，rdf）模式小鼠（簡稱rdf小鼠）。CSNB大鼠遺傳方式為性連鎖隱形遺傳，其突變基因?yàn)镃acna1f基因； rdf小鼠為常染色體隱性遺傳動(dòng)物模型，突變基因?yàn)镻de6b基因。本實(shí)驗(yàn)主要觀察CSNB模式大鼠出生后發(fā)育階段其視網(wǎng)膜中間神經(jīng)元之一水平細(xì)胞的變化，以及rdf模式小鼠出生后發(fā)育階段其視神經(jīng)髓鞘的變化，并探討其可能的機(jī)制，旨在為遺傳性視網(wǎng)膜疾病、視網(wǎng)膜視覺信號(hào)回路以及視覺神經(jīng)信號(hào)傳導(dǎo)的研究提供基本的實(shí)驗(yàn)依據(jù)。 方法 1.野生型SD大鼠及CSNB大鼠于出生后以隨機(jī)化原則按將要處死的時(shí)間點(diǎn)出生后15天（postnatal day15， PND15）、出生后30天（PND30）、出生后60天（PND60）分組。 2.應(yīng)用病理學(xué)染色方法、免疫組織化學(xué)染色方法、免疫組織熒光染色方法、全視網(wǎng)膜鋪片免疫熒光染色方法及western blotting方法觀察及對(duì)比野生型SD大鼠與CSNB大鼠出生后發(fā)育時(shí)期中其視網(wǎng)膜中水平細(xì)胞的變化。 3.野生型昆明種小鼠及rdf小鼠按照處理方式的不同（飼養(yǎng)于正常循環(huán)光照環(huán)境下及飼養(yǎng)于絕對(duì)暗室中）及將要處死的時(shí)間點(diǎn)（出生后7天、出生后14天、出生后28天，PND7、PND14、PND28）分組。按處理方式共分有4個(gè)組：野生型昆明小鼠對(duì)照組，rdf小鼠組，飼養(yǎng)于絕對(duì)暗室的野生型昆明小鼠對(duì)照組，飼養(yǎng)于絕對(duì)暗室的rdf小鼠組；其中各組內(nèi)又按處死時(shí)間分為PND7，PND14，PND28這3個(gè)時(shí)間點(diǎn)。并建立絕對(duì)避光暗室，將暗室組小鼠于出生當(dāng)天（PND0）即飼養(yǎng)于絕對(duì)暗室中。 4.應(yīng)用病理學(xué)染色方法及電子顯微鏡方法觀察野生型昆明種對(duì)照小鼠及rdf小鼠在出生后視覺發(fā)育時(shí)期中視神經(jīng)直徑、視神經(jīng)髓鞘有髓纖維數(shù)目、直徑及厚度的變化。 結(jié)果 1. CSNB大鼠的研究結(jié)果 CSNB大鼠在出生后15天、30天及60天（PND15,PND30，PND60）其視網(wǎng)膜中水平細(xì)胞數(shù)目及水平細(xì)胞在視網(wǎng)膜中的密度以及Calbindin蛋白的表達(dá)水平均低于同年齡組野生型SD大鼠（P 0.05）；野生型SD大鼠視網(wǎng)膜水平細(xì)胞數(shù)目及水平細(xì)胞的密度在各年齡時(shí)間點(diǎn)保持穩(wěn)定，而CSNB大鼠視網(wǎng)膜水平細(xì)胞數(shù)目及水平細(xì)胞的密度均隨著動(dòng)物年齡的增長呈減少趨勢(shì)；野生型SD大鼠視網(wǎng)膜中Calbindin蛋白的表達(dá)水平隨著動(dòng)物年齡的增長呈增加趨勢(shì)，而CSNB大鼠視網(wǎng)膜中Calbindin蛋白的表達(dá)水平在各年齡時(shí)間點(diǎn)沒有明顯的差異；CSNB大鼠水平細(xì)胞軸突較野生型SD大鼠稀疏；CSNB大鼠視網(wǎng)膜中內(nèi)核層與外叢狀層之間檢測(cè)到細(xì)胞凋亡；各組CSNB大鼠視網(wǎng)膜外叢狀層厚度均低于同年齡組野生型SD大鼠（P 0.05）。 2.視網(wǎng)膜退行性變和光線對(duì)小鼠視神經(jīng)髓鞘發(fā)育變化的影響結(jié)果 視神經(jīng)直徑：各組內(nèi)視神經(jīng)平均直徑隨年齡增長呈增加的趨勢(shì)。在出生后7天，，14天和28天（PND7, PND14和PND28），野生型昆明小鼠以及飼養(yǎng)于暗室中的野生型昆明小鼠在各時(shí)間點(diǎn)沒有明顯差異。rdf小鼠其視神經(jīng)直徑明顯小于同年齡組野生型昆明小鼠（P 0.05）。飼養(yǎng)于暗室中的rdf小鼠明顯大于同年齡組rdf小鼠（P 0.05），但是和同年齡組飼養(yǎng)于暗室中的野生型昆明小鼠比較沒有明顯差異。 有髓髓鞘數(shù)目：野生型昆明小鼠與同年齡組飼養(yǎng)于暗室中的野生型昆明小鼠相比顯著減少（P 0.05），rdf小鼠與同年齡組野生型昆明小鼠相比顯著增加（P 0.05）。飼養(yǎng)于暗室中的rdf小鼠與同年齡組飼養(yǎng)于暗室中的野生型昆明小鼠相比顯著減少（P 0.05）。 有髓髓鞘直徑：在PND14及PND28，野生型昆明小鼠顯著大于同年齡組飼養(yǎng)于暗室中的野生型昆明小鼠（P 0.05）。rdf組小鼠顯著小于同年齡組野生型昆明小鼠（P 0.05）。飼養(yǎng)于暗室中的rdf組小鼠顯著小于同年齡組rdf小鼠（P 0.05），而顯著大于同年齡組飼養(yǎng)于暗室中的野生型昆明小鼠（P 0.05）。 有髓髓鞘厚度：(1)小軸突組，rdf小鼠顯著大于同年齡組野生型昆明小鼠（P 0.05）。飼養(yǎng)于暗室中的野生型昆明小鼠顯著大于同年齡組昆明小鼠（P 0.05）。(2)大軸突組中，rdf小鼠顯著小于同年齡組野生型昆明小鼠（P 0.05）。飼養(yǎng)于暗室中的野生型昆明組小鼠顯著小于同年齡組昆明小鼠（P 0.05）。(3)小直徑組及大直徑組中，均發(fā)現(xiàn)在PND14d時(shí)，飼養(yǎng)于暗室中的rdf小鼠顯著小于同年齡組rdf小鼠（P 0.05），且顯著小于飼養(yǎng)于暗室中的野生型昆明小鼠（P 0.05），但是在PND28d時(shí)飼養(yǎng)于暗室中的rdf小鼠顯著大于同年齡組rdf小鼠（P 0.05），且顯著大于飼養(yǎng)于暗室中的野生型昆明小鼠（P 0.05）。 結(jié)論 通過本研究的實(shí)驗(yàn)發(fā)現(xiàn)，CSNB大鼠中水平細(xì)胞的數(shù)目與野生型SD大鼠相比減少，并且其數(shù)目隨著年齡的增長表現(xiàn)了下降的趨勢(shì)，外叢狀層厚度變薄，提示了先天性靜止性夜盲可以引起視網(wǎng)膜水平細(xì)胞缺失并且抑制視網(wǎng)膜水平細(xì)胞的發(fā)育。 小鼠中視神經(jīng)髓鞘的發(fā)育在一定程度上受到光剝奪和視網(wǎng)膜退行性變的獨(dú)立負(fù)性影響，但是光剝奪對(duì)rdf小鼠的視神經(jīng)髓鞘的抑制有一定的延緩作用，提示了光剝奪及視網(wǎng)膜退行性變各自都在一定程度上抑制了視神經(jīng)髓鞘的發(fā)育，但是發(fā)育階段的光剝奪卻可以在一定程度上延緩視網(wǎng)膜變性疾病視神經(jīng)髓鞘發(fā)育的改變。 本實(shí)驗(yàn)的研究結(jié)果提示，基因缺陷導(dǎo)致的遺傳性疾病在視覺發(fā)育期即可影響視覺回路的信號(hào)傳遞，不僅僅局限于疾病基因表達(dá)的靶組織和細(xì)胞結(jié)構(gòu)，由于視覺回路是一個(gè)復(fù)雜而精細(xì)的整體關(guān)聯(lián)的結(jié)構(gòu)，疾病也影響到視覺回路的其他部位，本實(shí)驗(yàn)的結(jié)果也進(jìn)一步提示到，在視覺發(fā)育期，是對(duì)疾病發(fā)展以及視覺回路信號(hào)傳遞的過程進(jìn)行干預(yù)和處理的關(guān)鍵時(shí)期，提示了在這一時(shí)期進(jìn)行治療時(shí)機(jī)可能更為關(guān)鍵并且治療效果可能更為顯著。
[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
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R774.1

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