本文選題：視網(wǎng)膜 + RCS大鼠　； 參考：《第三軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 視網(wǎng)膜色素變性(retinitis pigmentosa, RP)是一種由于感光細胞進行性缺失,導(dǎo)致視網(wǎng)膜內(nèi)各層神經(jīng)元發(fā)生繼發(fā)變性的嚴重致盲性疾病,近年來人們已對該病的治療進行了各種嘗試,包括視網(wǎng)膜移植、基因治療、視覺假體、藥物干預(yù)等,取得了一定的進展,但由于視網(wǎng)膜色素變性的發(fā)病機制尚不明確,因此為了明確其發(fā)病機制,尋找有效的治療手段,我們需要對該疾病進行深入的研究。 視網(wǎng)膜神經(jīng)節(jié)細胞(retinal ganglion cells, RGCs)是視網(wǎng)膜內(nèi)的第三級神經(jīng)元,也是視網(wǎng)膜發(fā)育中第一個產(chǎn)生的神經(jīng)元,其樹突主要接受視網(wǎng)膜內(nèi)的第二級神經(jīng)元視錐雙極細胞(retinal bipolar cells, RBCs)的信息傳遞,同時也可通過無長突細胞橫向聯(lián)系;其軸突延伸形成視神經(jīng),是視覺傳輸系統(tǒng)的重要組成部分。RGCs的樹突是接受神經(jīng)信號傳入的主要部位,樹突的形態(tài)發(fā)生對突觸信號的輸入、處理以及神經(jīng)環(huán)路的形成起重要作用。 在對慢性高眼壓、青光眼、年齡相關(guān)性黃斑變性和視網(wǎng)膜脫離等疾病的研究發(fā)現(xiàn),RGCs數(shù)量在病變過程中逐漸減少,殘留RGCs的樹突分支隨著病變的發(fā)展呈現(xiàn)遞減的趨勢。對多種視網(wǎng)膜變性動物模型的研究發(fā)現(xiàn),隨著感光細胞的死亡,RGCs雖然在數(shù)量上出現(xiàn)明顯減少,但是存活的RGCs形態(tài)并未發(fā)生改變并且仍然能誘發(fā)出動作電位。既往的研究對視網(wǎng)膜變性過程中RGCs形態(tài)是否發(fā)生改變意見不一。本實驗室既往的研究發(fā)現(xiàn),RCS大鼠(Royal college of surgeon rat, RCS)視網(wǎng)膜變性過程中,RGCs數(shù)量在早期有明顯減少,其中以胞體橫徑最大的α亞型減少最為顯著,但是這些存活的α亞型RGCs在視網(wǎng)膜變性過程中形態(tài)是否發(fā)生改變未作進一步研究。綜上所述,我們提出如下假設(shè):RCS大鼠視網(wǎng)膜變性過程中,隨著感光細胞的不斷變性死亡,視錐雙極細胞因失去信號輸入,其位于內(nèi)叢狀層的與RGCs進行突觸聯(lián)系的軸突分支發(fā)生了形態(tài)學(xué)改變,導(dǎo)致RGCs中對損傷最為敏感的α亞型RGCs在突觸形態(tài)上形態(tài)發(fā)生改變。 針對以上假設(shè),本文的主要研究內(nèi)容及研究結(jié)果如下: 1、通過對視網(wǎng)膜DiI染色方法進行改進,采用視網(wǎng)膜鋪片DiI示蹤標(biāo)記RGCs的方法,從單細胞水平對α亞型神經(jīng)節(jié)細胞樹突一二級分支數(shù)目、一級分支直徑及樹突分支頻率的變化進行研究,了解RCS大鼠視網(wǎng)膜變性過程中α亞型RGCs樹突的形態(tài)學(xué)變化。結(jié)果發(fā)現(xiàn):1)RCS大鼠視網(wǎng)膜變性過程中,P21dα亞型RGCs一級分支直徑出現(xiàn)顯著增粗,但是在病程的中晚期未見改變。α亞型RGCs一級分支直徑為何在RCS大鼠變性早期出現(xiàn)顯著增粗,這種改變有何意義尚不清楚,有待于進一步研究。2)RCS大鼠視網(wǎng)膜變性過程中,α亞型RGCs樹突分支頻率在早期P21、30d有顯著增加,而在晚期P60、90d時RGCs的樹突分支頻率顯著減少。樹突分支頻率是樹突發(fā)生分叉的次數(shù),反映了樹突數(shù)量的變化,因此,我們推測在RCS大鼠視網(wǎng)膜變性早期,隨著上級神經(jīng)元數(shù)量的減少,為了接受上級神經(jīng)元的信息傳遞并與周圍神經(jīng)元形成突觸聯(lián)系,α亞型RGCs代償性更早地伸出更多的樹突,與周圍的神經(jīng)元形成突觸聯(lián)系以維持其功能,在視網(wǎng)膜變性的中晚期,即P60、90d,隨著視網(wǎng)膜各級神經(jīng)元的進行性減少,RGCs上級神元大量缺失使神經(jīng)信息傳入減少,視網(wǎng)膜內(nèi)殘留的α亞型RGCs樹突分支也出現(xiàn)大量萎縮丟失,分支數(shù)目顯著減少。我們的研究既對α亞型RGCs的樹突一二級粗大分支數(shù)目進行了測量,也對樹突的終末分支頻率進行了觀察,結(jié)果表明,在視網(wǎng)膜變性過程中α亞型RGCs樹突粗大分支數(shù)目沒有明顯改變,而其終末的細小分支則出現(xiàn)了明顯的減少。提示了視網(wǎng)膜變性過程中RGCs樹突形態(tài)變化主要在終末分支,這種形態(tài)學(xué)的改變可能與上級神經(jīng)元視錐雙極細胞的軸突數(shù)目減少導(dǎo)致的傳入信息減少有關(guān)。 2、應(yīng)用免疫組織化學(xué)方法,采用Recoverin特異標(biāo)記2型及8型視錐雙極細胞,觀察RCS大鼠視網(wǎng)膜變性過程中視錐雙極細胞軸突密度的變化。結(jié)果發(fā)現(xiàn):RCS大鼠視網(wǎng)膜變性過程中,隨著視網(wǎng)膜外核層的感光細胞進行性缺失,Recoverin陽性視錐雙極細胞的軸突密度在P30d開始出現(xiàn)減少,P90d減少更為顯著。結(jié)果提示:失去上一級信號傳入后,視錐雙極細胞的數(shù)目和軸突分支出現(xiàn)減少,這種改變可能對RGCs的信息傳入產(chǎn)生影響。 綜上所述,本課題得出如下結(jié)論: 1、采用視網(wǎng)膜鋪片DiI熒光染色,可以較好的觀察神經(jīng)節(jié)細胞形態(tài),對了解和分析正常及病變中神經(jīng)節(jié)細胞形態(tài)特點及變化規(guī)律提供了一種簡單易行的方法。 2、RCS大鼠視網(wǎng)膜變性過程中,α亞型RGCs樹突一、二級分支數(shù)目無顯著變化,而樹突分支頻率在變性過程中呈現(xiàn)先增高后降低的趨勢。結(jié)果提示:RCS大鼠視網(wǎng)膜變性過程中,α亞型RGCs樹突形態(tài)的改變可能主要是其樹突終末分支的萎縮和丟失,而這種形態(tài)上的改變可能是由于傳入信息減少所致,但是其在功能上發(fā)生了怎樣的改變有待于進一步研究。 3、RCS大鼠視網(wǎng)膜變性過程中,2型和8型視錐雙極細胞軸突密度呈現(xiàn)逐漸遞減的趨勢。結(jié)果提示:在視網(wǎng)膜變性過程中,2型和8型視錐雙極細胞可能出現(xiàn)了細胞數(shù)量的減少,并且其軸突末梢分支也出現(xiàn)了萎縮和丟失,這種軸突形態(tài)上的改變可能對下級神經(jīng)元RGCs的信息傳入產(chǎn)生影響。
[Abstract]:Retinitis pigmentosa (RP) is a serious blinding disease causing secondary degeneration of the neurons in the retina due to the progressive loss of photoreceptor cells. In recent years, many attempts have been made for the treatment of the disease, including retinal transplantation, gene therapy, visual prosthesis, drug intervention and so on. Some progress, but the pathogenesis of retinitis pigmentosa is not clear, so in order to identify its pathogenesis and find effective treatment, we need to study the disease in depth.
The retinal ganglion cell (retinal ganglion cells (RGCs)) is the third stage neuron in the retina, the first neuron produced in the development of the retina, and its dendrite is mainly transmitted by the information transmission of the retinal bipolar cells (RBCs) in the retina of the retina, as well as through the transversal of the amacrine cells. The axon extends to the optic nerve. It is an important part of the visual transmission system, the dendrite of.RGCs is the main part of the nerve signal. The morphogenesis of the dendrite plays an important role in the input of the synaptic signal, the processing and the formation of the nerve loop.
In the study of chronic ocular hypertension, glaucoma, age-related macular degeneration and retinal detachment, the number of RGCs decreased gradually during the lesion, and the dendritic branching of the residual RGCs showed a decreasing trend with the development of the lesion. However, there is a significant decrease in number, but the surviving RGCs morphology does not change and still can induce action potential. Previous studies have disagreed on whether RGCs morphologic changes occurred during retinal degeneration. Previous studies in this laboratory found that RCS rats (Royal College of surgeon rat, RCS) were in the process of retinal degeneration. The number of RGCs decreased significantly in the early stage, among which the largest alpha subtype of the cytosolic transverse diameter decreased most significantly, but the morphologic changes of the surviving alpha subtype RGCs were not further studied in the retinal degeneration process. In summary, we put forward the following hypothesis: in RCS rat retinal degeneration process, with the continuous photoreceptor cells In denatured death, the axonal branch of the conical bipolar cell, which is located in the inner plexiform layer, has a synaptic connection with RGCs, resulting in the loss of signal input, resulting in a change in the morphology of the most sensitive RGCs subtype RGCs in the synapse.
In view of the above assumptions, the main research contents and results are as follows:
1, by improving the retinal DiI staining method and using the method of DiI tracer labeling RGCs, the changes of the number of branches of the dendritic one or two branches, the diameter of the primary branch and the frequency of the dendritic branching were studied from the single cell level to understand the morphology of the RGCs dendrites of the RCS subtype of the retina during the degeneration of the retina of the rat. The results are as follows: 1) in the process of retinal degeneration in RCS rats, the diameter of the primary branch of the P21d alpha subtype RGCs is markedly thickened, but it has not changed in the middle and late stages of the disease. Why is the primary branch diameter of the alpha subtype RGCs in the early stage of the denaturation of the RCS rat obviously thickening, what is the significance of this change is still unclear, and it needs to further study.2) RCS In the process of retinal degeneration, the frequency of the RGCs dendrite branch of the alpha subtype increased significantly in the early P21,30d, and the frequency of the dendritic branching decreased significantly at the late P60,90d. The frequency of the dendritic branching was the number of dendrites, reflecting the changes in the number of dendrites. Therefore, we speculate that in the early stage of retinal degeneration in RCS rats, the frequency of dendrites in the RCS rat was with the superior. The reduction in the number of neurons, in order to accept the information transmission of superior neurons and to form synaptic connections with the surrounding neurons, the alpha subtype RGCs compensatory to extend more dendrites earlier and form synaptic connections with the surrounding neurons in order to maintain its function. In the middle and late stages of retinal degeneration, P60,90d, with the progression of retinal neurons in the retina. The loss of RGCs superior deity resulted in the reduction of neural information, and a large number of atrophy and loss of the branches of the RGCs dendrites in the retina, and the number of branches decreased significantly. Our study not only measured the number of one or two grade coarse branches of the dendrites of the RGCs subtype, but also observed the terminal branch frequency of the dendrites. The results showed that the number of the coarse branches of the RGCs dendrite of the alpha subtype did not change obviously during the degeneration of retina, and the fine branches of the end were obviously reduced. It was suggested that the morphological changes of RGCs dendrites in the process of retinal degeneration were mainly in the terminal branch, which may be related to the axis of the pyramid bipolar cells of the superior neurons. The decrease of the number of inrush is associated with a decrease in the number of sudden inrush.
2, the changes in the axon density of cone bipolar cells in the retinal degeneration process of RCS rats were observed by Recoverin specific labeling of type 2 and type 8 conical bipolar cells. The results showed that in the degeneration process of RCS rats, the Recoverin positive cone bipolar disorder with the absence of the photoreceptor cells in the outer retinal nucleus. The cell axon density decreased at the beginning of P30d, and the P90d decreased more significantly. The results suggest that the number of cone bipolar cells and the axon branches decrease after the loss of the upper level signal, and this change may affect the information afferent of RGCs.
To sum up, the following conclusions are drawn.
1, the morphology of ganglion cells can be well observed by DiI fluorescent staining, which provides a simple and easy way to understand and analyze the morphological characteristics and changes of ganglion cells in normal and pathological changes.
2, in the process of retinal degeneration in RCS rats, the number of RGCs dendrites of alpha subtype has no significant change, but the branch frequency of the dendrite increases first and then decreases in the denaturation process. The results suggest that the modification of the RGCs dendritic morphology of the RCS subtype may be mainly the atrophy and loss of the end branch of the dendrite of the rat retina during the degeneration of retina. This change may be due to the reduction of incoming information, but its functional changes need further study.
3, during the retinal degeneration of RCS rats, the axon density of type 2 and 8 types of cone bipolar cells gradually diminishing. The results suggest that in the process of retinal degeneration, the number of cells in type 2 and 8 types of cone bipolar cells may decrease, and the axon branches are also atrophied and lost, and the morphological changes of this axon are changed. It may affect the incoming information of lower neurons RGCs.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R774.1

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