本文選題：RCS大鼠 + 視網(wǎng)膜ON型雙極細(xì)胞　； 參考：《第三軍醫(yī)大學(xué)》2010年博士論文

【摘要】：視網(wǎng)膜退行性變疾病是嚴(yán)重的致盲眼病,對(duì)于這類疾病目前尚缺乏有效阻止病變進(jìn)展和恢復(fù)視網(wǎng)膜功能的治療方法。其中,視網(wǎng)膜色素變性(Retinitis pigmentosa,RP)是一組以進(jìn)行性感光細(xì)胞及色素上皮功能喪失為共同表現(xiàn)的遺傳性疾病,是遺傳性視覺損害和盲目的最常見原因之一。近年來對(duì)視網(wǎng)膜色素變性疾病的臨床及基礎(chǔ)研究取得一定進(jìn)展,但視網(wǎng)膜色素變性的發(fā)病機(jī)制,及在病變過程中各種視網(wǎng)膜神經(jīng)細(xì)胞的病理改變及功能狀態(tài)尚不清楚。 視網(wǎng)膜雙極細(xì)胞是脊椎動(dòng)物視網(wǎng)膜的谷氨酸中間神經(jīng)元,接受光感受器細(xì)胞的信號(hào)輸入,在整合后傳遞至無長突細(xì)胞和神經(jīng)節(jié)細(xì)胞,雙極細(xì)胞在視網(wǎng)膜信號(hào)傳遞的過程處于一個(gè)關(guān)鍵的位置。在正常哺乳類動(dòng)物視網(wǎng)膜中視桿通路和視錐通路在外叢狀層相互獨(dú)立。視桿雙極細(xì)胞均為ON型,視錐雙極細(xì)胞分為ON型和OFF型,離子型谷氨酸能受體表達(dá)于OFF型雙極細(xì)胞胞膜上,ON型雙極細(xì)胞上以代謝性谷氨酸能受體為主,這兩種受體分別為不同亞型雙極細(xì)胞的主要功能性受體,對(duì)雙極細(xì)胞的功能起著重要的作用。 在多種視網(wǎng)膜變性動(dòng)物模型的研究中,業(yè)已發(fā)現(xiàn)在視網(wǎng)膜變性過程中,視網(wǎng)膜雙極細(xì)胞出現(xiàn)變構(gòu)的現(xiàn)象。這種變構(gòu)包括:1、雙極細(xì)胞在變性過程中失去樹突,其樹突上谷氨酸受體的表達(dá)出現(xiàn)了變化。2、雙極細(xì)胞能夠伸出新的突起與殘存的感光細(xì)胞形成異常連接。在針對(duì)以視錐細(xì)胞變性為主的視網(wǎng)膜變性動(dòng)物模型的研究中發(fā)現(xiàn),失去視錐感光細(xì)胞的視錐-雙極細(xì)胞能夠伸出突起與殘存的視桿感光細(xì)胞產(chǎn)生新的連接,在以視桿細(xì)胞變性為主的動(dòng)物模型中也有同樣的發(fā)現(xiàn),這些發(fā)現(xiàn)表明失去上一級(jí)信號(hào)傳入的雙極細(xì)胞在試圖尋找新的信號(hào)輸入。3、雙極細(xì)胞因?yàn)槟け砻娴鞍追肿拥淖兓憩F(xiàn)出功能特性的一些變化,例如Varela發(fā)現(xiàn)失去視桿信號(hào)傳入的視桿-雙極細(xì)胞呈現(xiàn)出對(duì)GABA更為強(qiáng)烈的反應(yīng);另有文獻(xiàn)表明,失去視桿細(xì)胞信號(hào)后的雙極細(xì)胞mGluR6的mRNA表達(dá)水平在變性晚期呈現(xiàn)明顯上升的趨勢,這種現(xiàn)象可以理解為是對(duì)谷氨酸能受體丟失的一種反應(yīng),同時(shí)Marc發(fā)現(xiàn)在變性過程中,視網(wǎng)膜表達(dá)代謝性谷氨酸能受體的雙極細(xì)胞減少,表達(dá)離子型谷氨酸能受體的雙極細(xì)胞增加,說明變性過程中雙極細(xì)胞的主要功能型受體的表達(dá)也發(fā)生了變化。而雙極細(xì)胞的這一切變構(gòu)特點(diǎn)會(huì)影響到雙極細(xì)胞膜學(xué)電生理特性。 在以視桿細(xì)胞變性為起始的視網(wǎng)膜變性動(dòng)物模型中,首先受到影響的就是失去視桿信號(hào)來源的視桿雙極細(xì)胞,在哺乳動(dòng)物中視桿雙極細(xì)胞均為ON型細(xì)胞,其類型相對(duì)于分型復(fù)雜的OFF型雙極細(xì)胞較為單一。根據(jù)既往的研究,我們推測殘存的視桿雙極細(xì)胞在失去視桿細(xì)胞的信號(hào)輸入后,會(huì)向其他殘存的感光細(xì)胞例如視錐細(xì)胞伸出異常突起,并從而導(dǎo)致自身形態(tài)學(xué)和膜學(xué)特性上的進(jìn)一步變化。我們現(xiàn)有的技術(shù)手段還不能精確和完整的顯示這種異常突起連接的形態(tài)和功能,并觀察其對(duì)雙極細(xì)胞的影響。但是通過雙極細(xì)胞形態(tài)和電學(xué)特性的研究,我們可以從側(cè)面嘗試了解這種異常連接帶來的變化,從而更多的了解網(wǎng)膜變性中ON型雙極細(xì)胞變構(gòu)的特性。 那么,在以視桿細(xì)胞變性為起始視網(wǎng)膜色素變性疾病發(fā)展過程中,ON型雙極細(xì)胞形態(tài)及膜學(xué)電生理特性究竟發(fā)生了怎樣的改變,這一切其至今未見研究報(bào)道,值得我們深入的研究。 綜上所訴,我們假設(shè)在以視桿細(xì)胞變性為起始的RCS大鼠動(dòng)物模型中,隨著感光細(xì)胞的不斷變性死亡,ON型雙極細(xì)胞因失去正常感光細(xì)胞的信號(hào)輸入后發(fā)生了變構(gòu),可能出現(xiàn)了和視錐細(xì)胞之間的異常連接,導(dǎo)致ON型雙極細(xì)胞出現(xiàn)了形態(tài)學(xué)以及電生理學(xué)特性的改變。 針對(duì)上述假說,本文的研究內(nèi)容和主要研究結(jié)果如下: 1、不同發(fā)育階段RCS鼠視網(wǎng)膜ON-RBCs形態(tài)學(xué)特征的研究。采用冰凍切片、免疫熒光化學(xué)方法特異性標(biāo)記RCS鼠視網(wǎng)膜ON-RBCs細(xì)胞,研究在視網(wǎng)膜變性發(fā)展過程中,ON-RBCs細(xì)胞的病理改變,并對(duì)大鼠的ON-RBCs進(jìn)行單細(xì)胞內(nèi)Lucifer Yellow染色以觀察單個(gè)雙極細(xì)胞形態(tài)的改變。結(jié)果發(fā)現(xiàn):1)RCS鼠視網(wǎng)膜色素變性過程中,ON-RBCs在總體數(shù)量上呈現(xiàn)下降趨勢,在P60d起顯著低于對(duì)照組大鼠。表明失去上一級(jí)信號(hào)傳入后,ON-RBCs不可避免的發(fā)生繼發(fā)性死亡,這屬于視網(wǎng)膜神經(jīng)層神經(jīng)元在變性過程中的變構(gòu)特征之一。2)RCS大鼠視網(wǎng)膜色素變性過程中,在視錐感光細(xì)胞集中的后極部網(wǎng)膜區(qū)域,在變性后期呈現(xiàn)出mGluR6陽性雙極細(xì)胞數(shù)量增加的趨勢。原因可能系有新的異常信號(hào)的傳入促進(jìn)了ON-RBC的存活,并促使其他區(qū)域的ON-RBCs的異位遷徙。3)RCS大鼠視網(wǎng)膜色素變性過程中,以視桿感光細(xì)胞為主的中周部網(wǎng)膜區(qū)域,ON-RBCs因?yàn)槭バ盘?hào)傳入,數(shù)量急劇下降,P60d起顯著低于對(duì)照組大鼠。證明了失去信號(hào)來源的雙極細(xì)胞繼發(fā)于感光細(xì)胞出現(xiàn)大量的變性死亡,間接證明了得到視錐信號(hào)支持的ON-RBCs能夠更好的存活。4)RCS大鼠視網(wǎng)膜色素變性過程中,樹突形態(tài)發(fā)育不良,軸突終末分支減少,細(xì)胞呈現(xiàn)幼稚狀態(tài),同時(shí)雙極細(xì)胞在視網(wǎng)膜變性過程中向不同方向伸出mGluR6表達(dá)陽性的異常突起。在變性晚期,這些異常突起糾纏成結(jié),并可見功能性突觸存在的證據(jù)。說明網(wǎng)膜變性過程中的雙極細(xì)胞為尋找新的信號(hào)傳入具有一定的突觸可塑性,這種可塑性現(xiàn)象為進(jìn)一步解釋視網(wǎng)膜變性特點(diǎn)以及開展視網(wǎng)膜拯救措施提供了新的依據(jù)。 2、RCS鼠視網(wǎng)膜變性過程中ON-RBCs細(xì)胞電生理學(xué)特性研究。采用在視網(wǎng)膜切片上對(duì)ON-RBCs細(xì)胞膜片鉗記錄的方法,對(duì)不同發(fā)育階段的RCS鼠ON-RBCs細(xì)胞進(jìn)行全細(xì)胞記錄,研究ON-RBCs細(xì)胞在視網(wǎng)膜變性發(fā)展過程中基本膜學(xué)特性、外向K+電流的性質(zhì)及大小的變化。結(jié)果發(fā)現(xiàn):1)RCS鼠視網(wǎng)膜變性過程中ON-RBCs細(xì)胞的靜息膜電位逐漸降低,輸入阻逐漸升高,均與對(duì)照組有顯著差異,說明RCS鼠視網(wǎng)膜ON-RBCs細(xì)胞膜逐漸去極化。RCS鼠視網(wǎng)膜變性過程中ON-RBCs細(xì)胞的膜電容逐漸增加,且顯著高于對(duì)照組,說明RCS鼠視網(wǎng)膜ON-RBCs細(xì)胞膜在面積和體積上發(fā)生了變化,2)在給予去極化脈沖時(shí),ON-RBCs細(xì)胞主要表現(xiàn)出外向的K+電流,能夠被TEA及阻斷。說明正常ON-RBCs細(xì)胞膜上離子通道以K+通道為主,以外向性鉀離子電流為主的特性。3)在視網(wǎng)膜變性過程中,ON-RBCs細(xì)胞外向K+電流增大,表現(xiàn)出異于對(duì)照組ON-RBCs的外向型鉀離子電流特性,說明在變性過程中ON-RBCs功能活動(dòng)特性發(fā)生了變化。4)RCS鼠P60d和P90d時(shí)K+電流密度顯著高于對(duì)照組(P0.05)。提示隨視網(wǎng)膜色素變性進(jìn)展,ON-RBCs細(xì)胞膜上單位面積K+電流增加,說明RCS鼠ON-RBCs細(xì)胞膜上K+通道類型和數(shù)量以及功能活性發(fā)生改變。5)RCS鼠ON-RBCs細(xì)胞K+通道I-V曲線的變化同樣符合其離子電流變化特性,即在視網(wǎng)膜變性過程中ON-RBCs細(xì)胞胞膜上K+離子通道活性的改變,因而相對(duì)于對(duì)照組,隨著膜電位的去極化,細(xì)胞膜K+電流升高更加明顯。6)RCS大鼠在P60和P90d時(shí),其ON-RBCs在去極化時(shí)表現(xiàn)出的外向型鉀離子電流幅值以及電流密度介于對(duì)照組ON-RBCs和OFF-RBCs之間,膜學(xué)特性與兩者均有顯著差異。 因此,我們得出以下結(jié)論: 1) RCS大鼠視網(wǎng)膜變性過程中,ON-RBCs在總體數(shù)量上呈現(xiàn)下降趨勢。表明失去上一級(jí)信號(hào)傳入后,ON-RBCs不可避免的發(fā)生繼發(fā)性的數(shù)量上的丟失;2)RCS大鼠視網(wǎng)膜變性過程中,周邊視桿細(xì)胞集中的中周部網(wǎng)膜區(qū)域,ON-RBCs因?yàn)槭バ盘?hào)傳入,數(shù)量急劇下降。證明了失去信號(hào)來源的ON-RBCs繼發(fā)于感光細(xì)胞的變性出現(xiàn)急劇的數(shù)量上的缺失;3)RCS大鼠視網(wǎng)膜色素變性過程中,在以視錐細(xì)胞集中的后極部視網(wǎng)膜區(qū)域,因?yàn)槿匀淮嬖谟懈泄饧?xì)胞來源的信號(hào)輸入,促進(jìn)了ON-RBC的存活,并可能吸引了其他視網(wǎng)膜區(qū)域的ON-RBCs的遷徙; 4)RCS大鼠視網(wǎng)膜色素變性過程中,樹突形態(tài)發(fā)育不良,軸突終末分支減少,細(xì)胞呈現(xiàn)幼稚狀態(tài),同時(shí)雙極細(xì)胞在網(wǎng)膜變性過程中向不同方向伸出mGluR6表達(dá)陽性的異常突起。在變性晚期,這些異常突起糾纏成結(jié),并可見功能性突觸存在的證據(jù)。說明變性網(wǎng)膜中的雙極細(xì)胞為尋找新的信號(hào)傳入具有一定的突觸可塑性;5)RCS鼠視網(wǎng)膜ON-RBCs細(xì)胞的離子通道特性主要表現(xiàn)為去極化時(shí)的外向K+電流。在視網(wǎng)膜變性進(jìn)程中,這種外向電流幅值均高于對(duì)照組,這揭示了ON-RBCs細(xì)胞在視網(wǎng)膜變性過程中功能狀態(tài)的變化,ON-RBCs可能通過此改變對(duì)抗K+在胞體內(nèi)中堆積,并反應(yīng)了ON-RBCs信號(hào)傳導(dǎo)功能的轉(zhuǎn)變;6)在視網(wǎng)膜變性過程中,RCS大鼠在P60和P90d,其ON-RBCs的被動(dòng)膜學(xué)特性以及在去極化時(shí)的外向型鉀離子電流幅值和電流密度與對(duì)照組ON-RBCs和OFF-RBCs兩者都不相似,存在顯著差異。說明RCS大鼠的ON-RBCs在視網(wǎng)膜變性過程中發(fā)生了細(xì)胞膜上K+通道類型和數(shù)量以及功能活性發(fā)生改變,這種變構(gòu)使其電生理特性異與任何一種類型的RBCs。
[Abstract]:In recent years , retinal pigment degeneration ( RP ) is one of the most common causes of hereditary visual impairment and blindness . The pathogenesis of retinal pigment degeneration and the pathological changes and functional states of various retinal neurons in the course of pathological changes are not clear .

The retinal bipolar cells are the intermediate neurons of glutamic acid in the retina of the vertebrate . The signal input of the photoreceptor cells is accepted , and the bipolar cells are separated from each other in the process of retinal signal transmission after integration . The bipolar cells in the normal mammalian retina are divided into the ON type and the OFF type . The two receptors are the main functional receptors of the bipolar cells of the OFF type . The two receptors play an important role in the function of bipolar cells .

In the study of various animal models of retinal degeneration , it has been found that , in the course of degeneration of retinal degeneration , bipolar cells lose dendritic cells , and the expression of glutamate receptors on their dendritic cells changes .

In the animal model of retinal degeneration with the degeneration of the optic rod as the starting material , it is first affected by the loss of the apparent pole bipolar cell of the apparent pole signal source , and the bipolar cell in the middle of the mammal is the ON type cell , and its type is relatively single relative to the typing complex OFF type bipolar cell . According to the previous research , we can not accurately and completely display the morphology and function of the abnormal protrusion connection , and observe its influence on the bipolar cell . However , we can try to understand the change of the abnormal connection from the side by studying the morphology and electrical characteristics of the bipolar cell .

So , in the course of the development of retinal pigment degeneration based on the degeneration of the optic cells , the morphology of the ON type bipolar cells and the electrophysiological characteristics of the membrane were changed .

In conclusion , we assume that in the animal model of RCS rats with the degeneration of the optic cells as the starting point , the ON - type bipolar cells have been altered due to the loss of the signal input of the normal photoreceptor , and the abnormal connection between the ON - type bipolar cells and the cone cells may occur , resulting in changes in morphology and electrophysiological properties of the ON - type bipolar cells .

According to the hypothesis , the research contents and main results of this paper are as follows :

In the course of degeneration of rat retinal pigment , ON - RBCs showed a decrease in the number of positive bipolar cells . In the course of degeneration of RCS rats , the number of ON - RBCs was significantly lower than that of the control rats .

In the process of retinal degeneration , the membrane capacitance of the ON - RBCs cells increased gradually , and the K + current in the membrane of the RCS rats was significantly higher than that of the control group ( P0.05 ) .

Therefore , we conclude that :

In the course of degeneration of the retinal degeneration of RCS rats , the changes of the function state of ON - RBCs in the peripheral optic cells were observed .
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R774.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張辰星,陰正勤,許紅霞,王仕軍,姚軍平;RCS大鼠病變發(fā)育過程中視網(wǎng)膜神經(jīng)節(jié)細(xì)胞形態(tài)學(xué)變化的研究[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2005年08期

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本文編號(hào)：2032253