【摘要】：第一部分N-亞硝基-N-甲基脲誘導(dǎo)小鼠視網(wǎng)膜變性病變模型的特征 目的：探討N-亞硝基-N-甲基脲(MNU)誘導(dǎo)的小鼠視網(wǎng)膜變性病變模型的形態(tài)、血管和電生理特征及其機(jī)制。 方法：32只5周齡的C57/BL小鼠隨機(jī)分為對照組和MNU造模組,造模組按60mg/kg腹腔注射給予N-亞硝基-N-甲基脲(MNU),對照組給予生理鹽水。在給藥后1d、2d、3d、5d、7d、2w、3w、4w和12w等不同時間點(diǎn)取視網(wǎng)膜標(biāo)本。行冰凍切片免疫熒光染色,觀察MNU誘導(dǎo)后光感受器細(xì)胞外節(jié)、雙極細(xì)胞、細(xì)胞突觸連接、神經(jīng)節(jié)等的形念變化,以及線粒體損傷和光感受器細(xì)胞氧化損傷的情況。用免疫印跡(Western blot)檢測蛋白表達(dá)水平。采用全視網(wǎng)膜鋪片免疫熒光染色,觀察神經(jīng)節(jié)細(xì)胞的數(shù)量和視網(wǎng)膜血管的形態(tài)。采用透射電鏡,觀察光感受器細(xì)胞的盤膜、光感受器細(xì)胞的線粒體、視網(wǎng)膜各層細(xì)胞核和帶狀突觸(synaptic ribbon)的超微結(jié)構(gòu)改變。采用Ganzfeld ERG檢測小鼠的全視網(wǎng)膜電生理改變。 結(jié)果：給予烷化劑藥物MNU后,采用免疫熒光染色。圖片顯示外核層(ONL)的厚度隨給藥后時間延長而逐漸減小,在一周內(nèi)減少到只剩下2-3個細(xì)胞核層,用OPN1SW抗體標(biāo)記光感受器細(xì)胞的外節(jié),顯示同樣外節(jié)的漸進(jìn)性丟失,提示位于視網(wǎng)膜外層的光感受器細(xì)胞對MNU引起的損傷最為敏感。10%的樣本出現(xiàn)后極部比周邊部的外節(jié)損傷更嚴(yán)重的現(xiàn)象。用蛋白激酶Ca (PKCa)抗體標(biāo)記雙極細(xì)胞,給藥后時間延長樹突呈現(xiàn)逐漸的回縮,突觸后致密蛋白95(PSD95)抗體標(biāo)記的光感受器細(xì)胞內(nèi)側(cè)端末梢也逐漸丟失,提示外叢狀層(OPL)的細(xì)胞間突觸連接受到破壞。膠質(zhì)纖維酸性蛋白(GFAP)抗體標(biāo)記的Muller細(xì)胞增生非常明顯,肥大的Muller細(xì)胞包裹受損的神經(jīng)元細(xì)胞,遍及整個視網(wǎng)膜細(xì)胞層,提示免疫機(jī)制被啟動。在外核層,與對照組相比,給MNU后, Nitrotyrosine抗體標(biāo)記的硝基酪氨酸和S-Nitroso-Cysteine抗體標(biāo)記的亞硝基化半胱氨酸的熒光信號明顯增多,提示MNU能導(dǎo)致外核層的氧化損傷。熱休克蛋白60(HSP60)是線粒體的常用標(biāo)記物,給MNU后,觀察到光感受器的線粒體逐漸丟失,給MNU后第3天,信號就完全消失；在內(nèi)核層,HSP60抗體標(biāo)記的線粒體熒光信號減少,但在給MNU后一周并未完全消失；線粒體損傷提示光感受器細(xì)胞的凋亡。無論是在免疫熒光切片還是鋪片上,Brn-3a抗體標(biāo)記的神經(jīng)節(jié)細(xì)胞的數(shù)量均并未見無明顯丟失。掃描電子顯微鏡顯示,給予藥物MNU后一周,光感受器細(xì)胞的核呈濃染色；給予MNU后3天內(nèi),光感受器細(xì)胞線粒體不同程度的腫脹,給MNU后5-7天,線粒體消失。給MNU后一周,光感受器下游的雙極細(xì)胞核周間隙增寬,在雙極細(xì)胞胞漿中有大量的自噬體。濃染核、線粒體腫脹和自噬體都是細(xì)胞凋亡的典型標(biāo)記。此外,還在電鏡下觀察了視網(wǎng)膜特有的帶狀突觸結(jié)構(gòu),在對照組的外叢狀層,觀察到大量典型的帶狀突觸,給藥MNU后第3天,數(shù)量大量減少,殘存的少量帶狀突觸其長度變短,邊緣模糊,周邊缺乏典型的水平細(xì)胞和雙極細(xì)胞的內(nèi)陷末梢結(jié)構(gòu)。在電鏡下,在給MNU后一周的樣本中,未觀察到內(nèi)叢狀層(IPL)和神經(jīng)節(jié)細(xì)胞層(GCL)的明顯變化。用Isolection IB6熒光染色顯示的視網(wǎng)膜血管結(jié)構(gòu),給MNU后,大分支血管壁結(jié)構(gòu)逐漸遭到破壞,末梢還出現(xiàn)大量血管叢樣結(jié)構(gòu)。Weastern Blot顯示GFAP表達(dá)水平在給MNU后一天即上升,7天達(dá)高峰；全視網(wǎng)膜的HSP60和細(xì)胞色素C氧化酶亞基IV (COX4)的水平下降約1/5,提示給MNU后一周內(nèi),光感受器的線粒體受到損傷,內(nèi)側(cè)其他視網(wǎng)膜細(xì)胞的線粒體尚存。給MNU后第3天,全視野ERG結(jié)果顯示：最大混合反應(yīng)的a波和b波波幅均明顯下降。 結(jié)論： MNU誘導(dǎo)模型主要導(dǎo)致光感受器細(xì)胞的丟失,并繼發(fā)視網(wǎng)膜神經(jīng)組織的結(jié)構(gòu)重塑,其凋亡的機(jī)制可能是通過氧化損傷,該模型為研究RP和干性AMD等視網(wǎng)膜變性性疾病提供了有價值的動物模型。第二部分大麻素受體1抑制劑SR141716A緩解MNU誘導(dǎo)視網(wǎng)膜變性病變模型的光感受器損傷 目的：研究大麻素受體1抑制劑SR141716A (SRI)緩解MNU誘導(dǎo)視網(wǎng)膜變性病變模型的光感受器損傷及機(jī)制。 方法：造模組按50mg/kg腹腔注射給予N-亞硝基-N-甲基脲(MNU),對照組給予生理鹽水和DMSO,干預(yù)組給于大麻素受體1和2的非特異性抑制劑WIN55,212-2(WIN)或SR1或SR144528(SR2).按照給藥的藥物成分,30只5周齡的C57/BL小鼠隨機(jī)分為以下七組：MNU組、WIN組、SR1組、MNU+WIN組、MNU+SR1組、MNU+WIN+SRl組MNU+WIN+SR2組,觀察給MNU后3天、5天、7天早中晚三個時期SR1對MNU造成的外核層損傷的影響。視網(wǎng)膜切片免疫熒光染色,顯示大麻素受體1(CB1)在視網(wǎng)膜各層的分布。免疫熒光雙標(biāo)法顯示CB1在視網(wǎng)膜各層細(xì)胞上的表達(dá)情況。急性分離細(xì)胞免疫熒光觀察單個雙極細(xì)胞上大麻素的表達(dá)。Real-time PCR定量觀察大麻素受體mRNA水平的表達(dá)。免疫熒光染色觀察給大麻素藥物后光感受器厚度變化和膠質(zhì)增生。全視網(wǎng)膜鋪片免疫熒光染色血管顯示血管損傷。薄片膜片鉗技術(shù)記錄ON型和OFF型雙極細(xì)胞的對各種藥物的電生理反應(yīng)。 結(jié)果：免疫熒光結(jié)果顯示CB1在視網(wǎng)膜是有表達(dá)的,CB1在視網(wǎng)膜外叢狀層和內(nèi)叢狀層的表達(dá)最為明顯。用抗體OPN1SW、PKC a、Brn3a和GFAP分別標(biāo)記光感受器細(xì)胞外節(jié)、雙極細(xì)胞、神經(jīng)節(jié)細(xì)胞的核和Muller細(xì)胞,分別與CB1抗體行免疫熒光雙標(biāo),提示CB1在光感受器細(xì)胞外節(jié)、內(nèi)核層雙極細(xì)胞的胞體和神經(jīng)節(jié)細(xì)胞的胞體也是有表達(dá)的,在Muller細(xì)胞上沒有表達(dá)。急性分離的雙極細(xì)胞用PKC α和CB抗體行免疫熒光雙標(biāo),證實了雙極細(xì)胞上的確有CB1的表達(dá). Real-time PCR結(jié)果顯示：MNU損傷后,大麻素受體1的基因表達(dá)量無變化,大麻素受體2基因表達(dá)上調(diào)。單獨(dú)給WIN或WIN+SR1或WIN+SR2干預(yù)沒有改善作用。單獨(dú)給SR1干預(yù)有改善作用。與MNU組比較,MNU+SR1的外核層厚度更厚,膠質(zhì)增生不明顯。全視網(wǎng)膜鋪片免疫熒光染色血管顯示：MNU+SR1組的大血管損傷輕,只見少量的異常末梢血管叢。薄片膜片鉗技術(shù)記錄：ON型視桿細(xì)胞對MNU的損傷比OFF型雙極細(xì)胞更敏感。SR1能部分緩解MNU誘導(dǎo)的光感受器損傷和及其對下游雙極細(xì)胞的影響。 結(jié)論：SR1能緩解MNU誘導(dǎo)視網(wǎng)膜外層變性模型的光感受器損傷。
[Abstract]:Part one: characteristics of N- nitroso -N- methylurea induced retinal degeneration in mice
AIM: To investigate the morphological, vascular and electrophysiological characteristics and mechanism of retinal degeneration induced by N-nitroso-N-methylurea (MNU) in mice.
Methods: Thirty-two five-week-old C57/BL mice were randomly divided into control group and MNU model group. The model group was given N-nitroso-N-methylurea (MNU) by intraperitoneal injection of 60mg/kg, and the control group was given normal saline. Retinal specimens were taken at different time points 1, 2, 3, 5, 7, 2, 3, 4 and 12 weeks after administration. The morphological changes of photoreceptor extracellular ganglion, bipolar cells, synaptic junction, ganglion, mitochondrial damage and oxidative damage of photoreceptor cells were observed. The expression of protein was detected by Western blot. The number of ganglion cells and retinal vessels were observed by panretinal immunofluorescence staining. Morphology. The ultrastructural changes of the disc membrane, mitochondria, nucleus and synaptic ribbon of photoreceptor cells were observed by transmission electron microscopy. The electrophysiological changes of the whole retina in mice were detected by Ganzfeld ERG.
RESULTS: Immunofluorescence staining was used after the alkylating agent MNU was given. The picture showed that the thickness of the outer nucleus layer (ONL) decreased gradually with the prolongation of administration time, and decreased to only 2-3 nucleus layers left in one week. The outer segment of photoreceptor cells was labeled with OPN1SW antibody, indicating gradual loss of the same outer segment of retina. In 10% of the samples, the posterior pole was more severely damaged than the peripheral segment. The dendrites of the bipolar cells labeled with protein kinase Ca (PKCa) antibody gradually retracted after administration, and the lateral end of the photoreceptor cells labeled with postsynaptic dendrin 95 (PSD95) antibody was observed. The gradual loss of peripherals suggests that the synaptic connections between the outer plexiform layer (OPL) cells are destroyed. The proliferation of GFAP antibody-labeled Muller cells is very obvious. Hypertrophic Muller cells encapsulate damaged neurons throughout the retinal cell layer, suggesting that the immune mechanism is activated. Compared with MNU, Nitrotyrosine labeled with Nitrotyrosine antibody and S-Nitroso-Cysteine labeled with nitrocysteine fluorescence signal increased significantly, suggesting that MNU can cause oxidative damage in the outer nuclear layer. Heat shock protein 60 (HSP60) is a common marker of mitochondria. After MNU is given, mitochondria of photoreceptors are gradually lost. On the 3rd day after MNU, the signal disappeared completely; on the inner nuclear layer, the mitochondrial fluorescence signal labeled by HSP60 antibody decreased, but did not disappear completely one week after MNU was given; mitochondrial damage indicated apoptosis of photoreceptor cells. Scanning electron microscopy showed that the nuclei of photoreceptor cells were stained intensively one week after administration of MNU. Within three days after administration of MNU, the mitochondria of photoreceptor cells were swollen in varying degrees, and disappeared 5-7 days after administration of MNU. In addition, the banded synaptic structures of the retina were observed under electron microscopy. In the outer plexiform layer of the control group, a large number of typical banded synapses were observed. On the third day after the administration of MNU, the number of banded synapses decreased significantly, and a small number of banded synapses remained. There were no obvious changes in the inner plexiform layer (IPL) and ganglion cell layer (GCL) in the specimens given MNU for one week under electron microscopy. Weastern Blot showed that the expression of GFAP increased one day after MNU administration and reached its peak on seven days. The levels of HSP60 and cytochrome C oxidase subunit IV (COX4) in the whole retina decreased by about one fifth, suggesting that the mitochondria of the photoreceptors were damaged within one week after MNU administration. Mitochondria remained in his retinal cells. Three days after MNU administration, full-field ERG showed that the amplitudes of a and B waves of the maximal mixed reaction decreased significantly.
CONCLUSION: The MNU-induced model mainly leads to the loss of photoreceptor cells and secondary structural remodeling of retinal nerve tissue. The mechanism of apoptosis may be through oxidative damage. This model provides a valuable animal model for the study of retinal degenerative diseases such as RP and dry AMD. Part II Cannabinoid receptor 1 inhibitor SR141716A slows down. Photoreceptor injury in MNU induced retinal degeneration model
AIM: To investigate the mechanism of cannabinoid receptor 1 inhibitor SR141716A (SRI) in alleviating photoreceptor damage in MNU-induced retinal degeneration.
METHODS: The model group was given N-nitroso-N-methylurea (MNU) by intraperitoneal injection of 50mg/kg, the control group was given normal saline and DMSO, and the intervention group was given non-specific inhibitors of cannabinoid receptor 1 and 2, WIN55, 212-2 (WIN) or SR1 or SR144528 (SR2). MNU+WIN+SR2 group, MNU+WIN group, MNU+SR1 group, MNU+WIN+SRl group and MNU+WIN+SR2 group were used to observe the effect of SR1 on the damage of outer nuclear layer induced by MNU at 3 days, 5 days, 7 days in the morning, middle and late stages. The expression of cannabinoid on single bipolar cells was observed by immunofluorescence assay. The expression of cannabinoid receptor mRNA was quantitatively detected by Real-time PCR. The changes of photoreceptor thickness and glial hyperplasia were observed by immunofluorescence staining after cannabinoid administration. Wound patch clamp technique was used to record the electrophysiological responses of ON and OFF bipolar cells to various drugs.
Results: Immunofluorescence showed that CB1 was expressed in the retina, especially in the outer plexiform layer and inner plexiform layer. Antibodies OPN1SW, PKC a, Brn3a and GFAP were used to label extracellular segment, bipolar cell, ganglion cell nucleus and Muller cell of photoreceptor, respectively, and immunofluorescence double labeling was performed with CB1 antibody. The expression of CB1 was confirmed by immunofluorescence double labeling of acute isolated bipolar cells with PKC-a and CB antibodies. Real-time PCR results showed that CB1 was expressed on the bipolar cells after MNU injury. Compared with MNU group, the outer nuclear layer of MNU + SR1 was thicker and the glial hyperplasia was not obvious. The whole retina slice immunofluorescence staining showed that MNU + SR1 group had a thicker outer nuclear layer and no obvious glial hyperplasia. Slice patch clamp technique showed that ON rod cells were more sensitive to MNU damage than OFF bipolar cells. SR1 partially alleviated MNU-induced photoreceptor damage and its effect on downstream bipolar cells.
Conclusion: SR1 can alleviate photoreceptor injury in MNU induced retinal degeneration.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R774.1

【共引文獻(xiàn)】

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