本文選題：斑馬魚 + 視網(wǎng)膜��； 參考：《吉林大學》2013年博士論文

【摘要】：視網(wǎng)膜色素變性是一種遺傳性致盲眼病，目前全世界范圍內(nèi)發(fā)病率為19-27/100，000，視網(wǎng)膜光感受器細胞進行性死亡是導致視力喪失的主要原因。光感受器細胞是終末分化的神經(jīng)元，它們不能再生，一旦喪失就無法替代。因而關于視網(wǎng)膜光感受器細胞退變機制的研究非常重要。 自1981年美國俄勒岡大學著名遺傳學家George streisinger關于斑馬魚人工雌核發(fā)育的研究在《Nature》發(fā)表后，斑馬魚作為研究遺傳發(fā)育的實驗動物模型開始引起科學家的極大關注。目前斑馬魚已成為最受重視的脊椎動物發(fā)育生物學模型之一。斑馬魚的視網(wǎng)膜感光細胞以視錐細胞為主，在一些視網(wǎng)膜疾病的研究上具有獨特優(yōu)勢，比如視網(wǎng)膜色素變性、年齡相關性黃斑變性等，選用斑馬魚作為模式動物極佳。 Crumbs蛋白復合物是上皮極性的一個關鍵調(diào)節(jié)物。Crumbs蛋白復合物高度保守，這個復合物中主要的成員-Crumbs(Crb)最初在黑腹果蠅中被發(fā)現(xiàn)，對果蠅正常的光感受器細胞形態(tài)形成及粘著小帶的形成和維持十分重要。在果蠅中只有一種Crb，在脊椎動物中有三種CRB-CRB1、CRB2和CRB3。 CRB1基因突變與人類多種常染色體隱性遺傳性視網(wǎng)膜變性相關，包括視網(wǎng)膜色素變性和Leber氏先天性黑蒙。目前對CRB1突變體尚未發(fā)現(xiàn)明確的基因型-表型之間的關聯(lián)，說明Crumbs蛋白復合物的其它成分可能也影響疾病的嚴重程度。 斑馬魚有5種Crb，它們分別是Crb1、Crb2a、Crb2b、Crb3a和Crb3b。在斑馬魚中，兩個Crb2基因(Crb2a和Crb2b)與視網(wǎng)膜的發(fā)育和形態(tài)學缺陷相關。Crb2b主要在斑馬魚視網(wǎng)膜視錐細胞內(nèi)節(jié)表達，Crb2b的細胞外區(qū)域介導細胞間粘附。Crb2b在維持光感受器細胞層完整性及穩(wěn)定視錐細胞馬賽克結構平面構建上發(fā)揮重要的作用。斑馬魚Crb2b功能與人類CRB1功能相似，研究Crb2b對斑馬魚光感受器細胞的影響有助于進一步揭示視網(wǎng)膜色素變性等退變性視網(wǎng)膜病變的發(fā)病機制。 精確地計數(shù)視網(wǎng)膜細胞在時間和空間上的分布是研究視網(wǎng)膜發(fā)育及退變的基礎。每個視網(wǎng)膜細胞只有一個細胞核，因此最直接計數(shù)視網(wǎng)膜細胞的方法是在光學顯微鏡下計數(shù)細胞核的數(shù)目。本論文首先結合JB-4塑膠樹脂包埋和福爾根染色的方法觀察斑馬魚視網(wǎng)膜細胞核。通過分析JB-4切片厚度、鹽酸水解程度及Schiff反應條件對細胞核染色的影響，尋找一個最優(yōu)地能夠敏感且特異染色斑馬魚視網(wǎng)膜細胞核的方法。實驗結果顯示：鹽酸濃度越高、浸潤時間越長，染色程度越強；鹽酸濃度越低、浸潤時間越短，染色程度越弱。低濃度的鹽酸可以通過延長浸潤時間而提高染色強度。染色強度隨著Schiff浸潤時間的延長而增加，2小時達到最強染色。在2μm組織切片中單個細胞核比在3μm組織切片、4μm組織切片及6μm組織切片中顯示出更清晰的核邊界。在2μm厚經(jīng)福爾根染色的JB-4切片中，視錐細胞中的常染色質(zhì)區(qū)域能夠清晰地和異染色質(zhì)區(qū)域區(qū)分開。與亞甲天藍II和核固紅染色相比，福爾根染色特異性和穩(wěn)定性更強。福爾根染色可以和標本包埋原位雜交技術相兼容。通過實驗結果分析發(fā)現(xiàn)了一個最優(yōu)地能夠敏感且特異染色斑馬魚視網(wǎng)膜細胞核的方法。這個簡單可靠的染色方法可以用于細胞計數(shù)及檢測視錐細胞染色質(zhì)整體結構，與標本包埋原位雜交技術相結合可同時觀察細胞核和mRNA表達。 本論文其次通過計數(shù)成年野生型斑馬魚視網(wǎng)膜神經(jīng)節(jié)細胞、內(nèi)核層細胞、水平細胞、視錐細胞及視桿細胞在視網(wǎng)膜不同區(qū)域內(nèi)的細胞數(shù)目，通過比較不同年齡斑馬魚之間相同視網(wǎng)膜區(qū)域細胞數(shù)目的變化及某一特定年齡斑馬魚不同視網(wǎng)膜區(qū)域之間細胞數(shù)目的變化探討野生型斑馬魚視網(wǎng)膜發(fā)育的特點。實驗結果顯示：野生型斑馬魚視網(wǎng)膜神經(jīng)節(jié)細胞、內(nèi)核層細胞、水平細胞、視桿細胞及視錐細胞在視網(wǎng)膜各個區(qū)域排列規(guī)整。斑馬魚早期視網(wǎng)膜各類細胞發(fā)育從周邊部開始，逐漸向中央及兩側(cè)延伸，最終上形成特殊的空間分布特點：除視桿細胞外各類細胞視網(wǎng)膜中心區(qū)細胞數(shù)目最多，向兩側(cè)逐漸減少形成正態(tài)分布；視桿細胞視網(wǎng)膜前部-后部方向后部旁中心區(qū)細胞數(shù)目最多，向兩側(cè)逐漸減少；腹部-背部方向背部旁中心區(qū)細胞數(shù)目最多，向兩側(cè)逐漸減少形成偏正態(tài)分布。野生型斑馬魚視網(wǎng)膜細胞在時間上有一定的分布規(guī)律：伴隨著斑馬魚的年齡增長，視網(wǎng)膜細胞總體呈現(xiàn)下降趨勢，視桿細胞在斑馬魚生長早期局部區(qū)域呈現(xiàn)上升趨勢。該實驗結果為進一步揭示視網(wǎng)膜發(fā)育機制及研究退變性視網(wǎng)膜病變的發(fā)病機制提供必要的基礎數(shù)據(jù)。另外，本實驗結果顯示斑馬魚隨年齡增長光感受器細胞整體呈現(xiàn)下降趨勢，晚期下降最為明顯。人類年齡相關性黃斑變性是隨年齡增長而進展的的光感受器細胞退變性疾病。通過本研究視網(wǎng)膜視錐細胞隨年齡增長逐漸死亡，特別是到晚期視錐細胞大量死亡提供的基礎數(shù)據(jù)可進一步揭示年齡相關性性黃斑變性的發(fā)病機制。 本論文最后計數(shù)兩個不同年齡階段pt108b成年斑馬魚視網(wǎng)膜不同區(qū)域細胞的數(shù)目，觀察pt108b斑馬魚視網(wǎng)膜細胞的空間分布；并通過與同年齡野生型斑馬魚相同視網(wǎng)膜區(qū)域細胞數(shù)目及視網(wǎng)膜細胞空間分布比較探討Crb2b對視網(wǎng)膜光感受器細胞的影響。實驗結果顯示：pt108b轉(zhuǎn)基因斑馬魚視網(wǎng)膜各類細胞在空間上并無像野生型斑馬魚一樣規(guī)律的分布。pt108b轉(zhuǎn)基因斑馬魚視網(wǎng)膜各類細胞分層破壞，細胞間排列松散，視錐細胞排列極其稀疏；特別是生長晚期細胞排列紊亂，個別區(qū)域視錐細胞完全喪失。pt108b轉(zhuǎn)基因斑馬魚不同時期視網(wǎng)膜各個區(qū)域視錐細胞數(shù)目較同齡野生型斑馬魚均顯著下降；且視網(wǎng)膜視錐細胞數(shù)目隨年齡增長顯著下降，至晚期視錐細胞數(shù)目急劇減少，個別區(qū)域視錐細胞幾乎完全消失。但生長晚期除視錐細胞外其它細胞數(shù)目在視網(wǎng)膜局部區(qū)域呈現(xiàn)不同程度的增多，，特別是視桿細胞在視網(wǎng)膜絕大多數(shù)區(qū)域均顯著增多。實驗結果說明Crb2b是維持成年斑馬魚視網(wǎng)膜各類細胞正常空間分布及視錐細胞存活的關鍵蛋白。Crb2b功能抑制可以導致成年斑馬魚視網(wǎng)膜視錐細胞死亡特別是生長晚期視錐細胞大量死亡以及神經(jīng)節(jié)細胞、內(nèi)核層細胞、水平細胞及視桿細胞在晚期視網(wǎng)膜局部區(qū)域不同程度的增生。pt108b轉(zhuǎn)基因斑馬魚由于Crb2b功能抑制可以導致視網(wǎng)膜視錐細胞進行性死亡，逐漸進展的光感受器細胞死亡是遺傳性視網(wǎng)膜色素變性顯著的特征。該動物模型有助于進一步深入研究視網(wǎng)膜色素變性的發(fā)病機制。
[Abstract]:Retinitis pigmentosa is a hereditary blinding ophthalmopathy. The incidence of the disease is 19-27/100000 worldwide. The progressive death of the retina photoreceptor cells is the main cause of visual loss. The photoreceptor cells are terminally differentiated neurons. They can not be regenerated and can not be replaced once they are lost. Therefore, the retinal light is not replaceable. The research on the mechanism of receptor cell degeneration is very important.
Since 1981, George Streisinger, a famous geneticist at the University of Oregon, published a study on zebrafish artificial ethe development of zebrafish, the zebrafish as an experimental animal model for genetic development began to arouse great attention of scientists. At present zebrafish has become one of the most important biological models for vertebrate development. The retina photosensitive cells of zebrafish are dominated by cone cells and have unique advantages in the study of some retinal diseases, such as retinitis pigmentosa, age related macular degeneration, and the selection of zebrafish as a model animal.
The Crumbs protein complex is a key regulator of the epithelial polarity, a key regulator of the.Crumbs protein complex, which is highly conserved. The main member of the complex, -Crumbs (Crb), was first found in the Drosophila melanogaster, which is important for the formation and maintenance of the normal photoreceptor cell morphology and the formation and maintenance of the sticky band. In the Drosophila, there is only one Crb in the fruit fly. There are three kinds of CRB-CRB1, CRB2 and CRB3. in vertebrates
The CRB1 gene mutation is associated with a variety of autosomal recessive retinal degeneration, including retinitis pigmentosa and Leber's congenital melanoma. There is no clear genotype phenotype association for CRB1 mutants, indicating that other components of the Crumbs protein complex may also affect the severity of the disease.
Zebrafish has 5 kinds of Crb, which are Crb1, Crb2a, Crb2b, Crb3a and Crb3b. in zebrafish. The two Crb2 genes (Crb2a and Crb2b) are associated with the retinal development and morphological defects..Crb2b is mainly expressed in the retina of the retina of the zebrafish retina. The extracellular domain mediates the intercellular adhesion in the maintenance of the photoreceptor cell layer. The Crb2b function of zebrafish is similar to that of human CRB1. The study of the effect of Crb2b on the photoreceptor cells of zebrafish can help to further reveal the pathogenesis of retinitis pigmentosa, such as retinitis pigmentosa.
Accurately counting the distribution of retinal cells in time and space is the basis for studying retinal development and degeneration. Each retinal cell has only one nucleus, so the most direct method of counting the retinal cells is to count the number of nuclei under an optical microscope. This paper first combined with JB-4 plastic resin embedding and formaldehyde staining. The effects of JB-4 slice thickness, hydrochloric acid hydrolysis degree and Schiff reaction condition on the cell nucleus staining were analyzed. The optimal method for sensitive and specific staining of zebrafish retinal nuclei was found. The results showed that the higher the concentration of salt acid, the longer the infiltration time, the degree of dyeing. The stronger the concentration, the lower the concentration of hydrochloric acid, the shorter the infiltration time, the weaker the dyeing degree. The low concentration of hydrochloric acid can increase the dyeing strength by prolonging the time of infiltration. The intensity of dyeing increases with the prolongation of the time of Schiff infiltration, and the strongest dyeing is reached at 2 hours. The single cell nucleus of the tissue section of 2 mu is compared to 3 mu m, 4 mu m tissue section and 6 mu. The M tissue section shows a clearer nuclear boundary. In the JB-4 section stained by the 2 m thick, the normal chromatin region in the cone cells can be clearly separated from the heterochromatin region. An optimal method for sensitive and specific staining of zebrafish retinal nuclei is found through experimental results. This simple and reliable staining method can be used for cell counting and detection of the overall structure of the cone cell chromatin, combined with the embedded in situ hybridization technique. The expression of nucleus and mRNA.
This thesis is followed by counting the number of cells in the retinal ganglion cells, core layer cells, horizontal cells, cone cells and rod cells in different retina of the retina of adult wild zebrafish, by comparing the changes in the number of cells in the same retina region between different age zebrafish and the different viewing nets of a certain age of a certain age zebrafish. The retinal ganglion cells of wild zebrafish, core layer cells, horizontal cells, rod cells and cone cells arranged regularly in various regions of the retina, and the development of various cells in the early retina of zebrafish from the periphery of the wild zebrafish. At the beginning, it gradually extends to the central and both sides, and finally forms a special spatial distribution feature: the number of cells in the central area of retina of various cells except the rod cells is the most, and the normal distribution is gradually reduced to both sides; the number of cells in the center of the posterior part of the retina of the rod cells is the most, and the abdomen decreases gradually to both sides. In the back, the number of cells in the central region of the back is the most, and the distribution of the retinal cells in the wild type zebrafish has a certain distribution rule. With the age of zebrafish growth, the retinal cells generally decline, and the rod cell is up in the early local area of the zebrafish. The experimental results provide necessary basic data to further reveal the mechanism of retinal development and the pathogenesis of degenerative retinopathy. In addition, the results of this experiment show that the overall decline of the photoreceptor cells in zebrafish with age increases, and the late decline is most obvious. The progressive death of retinal cone cells with age, especially the basic data provided by the massive death of advanced cone cells in this study, can further reveal the pathogenesis of age-related macular degeneration.
At the end of this paper, the number of cells in different regions of pt108b adult zebrafish pt108b was counted and the spatial distribution of pt108b zebrafish retina cells was observed. The photoreceptor of the retina was compared with the number of cells in the same retina region and the spatial distribution of retinal cells in the same age of the wild zebrafish of the same age. The experimental results showed that the pt108b transgenic zebrafish retina had no spatial distribution of various cells in the retina of the retina like wild zebrafish. The cells in the retina of the.Pt108b transgenic zebrafish were delaminated, the cells were loosely arranged and the cones were very sparse, especially in the late growth of the cells. The number of cone cells in each region of the retina of.Pt108b transgenic zebrafish was significantly lower than that of the same age wild zebrafish, and the number of retinal cone cells decreased significantly with age, and the number of late cone cells decreased dramatically, and the cone cells in a few regions almost completely disappeared. But the number of other cells outside the cone cells in the late growth stage increased in different degrees in the retina, especially in the vast majority of the retina. The results showed that Crb2b was the key protein.Crb2b to maintain the normal spatial distribution of various cells in adult zebrafish retina and the survival of the cones. Functional inhibition can lead to the death of retinal cone cells in adult zebrafish, especially in late growing cones, as well as ganglion cells, core layer cells, horizontal cells, and rod cells in different degrees in the local area of the late retina,.Pt108b transgenic zebra fish can lead to retina due to the inhibition of Crb2b function. The progressive death of the photoreceptor cells is a significant feature of hereditary retinitis pigmentosa. This animal model is helpful to further study the pathogenesis of retinitis pigmentosa.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R774.1

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