大麻素受体在视网膜外层变性模型中的神经保护作用及机制研究

发布时间：2018-08-27 05:44

【摘要】：第一部分N-亚硝基-N-甲基脲诱导小鼠视网膜变性病变模型的特征目的：探讨N-亚硝基-N-甲基脲(MNU)诱导的小鼠视网膜变性病变模型的形态、血管和电生理特征及其机制。方法：32只5周龄的C57/BL小鼠随机分为对照组和MNU造模组,造模组按60mg/kg腹腔注射给予N-亚硝基-N-甲基脲(MNU),对照组给予生理盐水。在给药后1d、2d、3d、5d、7d、2w、3w、4w和12w等不同时间点取视网膜标本。行冰冻切片免疫荧光染色,观察MNU诱导后光感受器细胞外节、双极细胞、细胞突触连接、神经节等的形念变化,以及线粒体损伤和光感受器细胞氧化损伤的情况。用免疫印迹(Western blot)检测蛋白表达水平。采用全视网膜铺片免疫荧光染色,观察神经节细胞的数量和视网膜血管的形态。采用透射电镜,观察光感受器细胞的盘膜、光感受器细胞的线粒体、视网膜各层细胞核和带状突触(synaptic ribbon)的超微结构改变。采用Ganzfeld ERG检测小鼠的全视网膜电生理改变。结果：给予烷化剂药物MNU后,采用免疫荧光染色。图片显示外核层(ONL)的厚度随给药后时间延长而逐渐减小,在一周内减少到只剩下2-3个细胞核层,用OPN1SW抗体标记光感受器细胞的外节,显示同样外节的渐进性丢失,提示位于视网膜外层的光感受器细胞对MNU引起的损伤最为敏感。10%的样本出现后极部比周边部的外节损伤更严重的现象。用蛋白激酶Ca (PKCa)抗体标记双极细胞,给药后时间延长树突呈现逐渐的回缩,突触后致密蛋白95(PSD95)抗体标记的光感受器细胞内侧端末梢也逐渐丢失,提示外丛状层(OPL)的细胞间突触连接受到破坏。胶质纤维酸性蛋白(GFAP)抗体标记的Muller细胞增生非常明显,肥大的Muller细胞包裹受损的神经元细胞,遍及整个视网膜细胞层,提示免疫机制被启动。在外核层,与对照组相比,给MNU后, Nitrotyrosine抗体标记的硝基酪氨酸和S-Nitroso-Cysteine抗体标记的亚硝基化半胱氨酸的荧光信号明显增多,提示MNU能导致外核层的氧化损伤。热休克蛋白60(HSP60)是线粒体的常用标记物,给MNU后,观察到光感受器的线粒体逐渐丢失,给MNU后第3天,信号就完全消失；在内核层,HSP60抗体标记的线粒体荧光信号减少,但在给MNU后一周并未完全消失；线粒体损伤提示光感受器细胞的凋亡。无论是在免疫荧光切片还是铺片上,Brn-3a抗体标记的神经节细胞的数量均并未见无明显丢失。扫描电子显微镜显示,给予药物MNU后一周,光感受器细胞的核呈浓染色；给予MNU后3天内,光感受器细胞线粒体不同程度的肿胀,给MNU后5-7天,线粒体消失。给MNU后一周,光感受器下游的双极细胞核周间隙增宽,在双极细胞胞浆中有大量的自噬体。浓染核、线粒体肿胀和自噬体都是细胞凋亡的典型标记。此外,还在电镜下观察了视网膜特有的带状突触结构,在对照组的外丛状层,观察到大量典型的带状突触,给药MNU后第3天,数量大量减少,残存的少量带状突触其长度变短,边缘模糊,周边缺乏典型的水平细胞和双极细胞的内陷末梢结构。在电镜下,在给MNU后一周的样本中,未观察到内丛状层(IPL)和神经节细胞层(GCL)的明显变化。用Isolection IB6荧光染色显示的视网膜血管结构,给MNU后,大分支血管壁结构逐渐遭到破坏,末梢还出现大量血管丛样结构。Weastern Blot显示GFAP表达水平在给MNU后一天即上升,7天达高峰；全视网膜的HSP60和细胞色素C氧化酶亚基IV (COX4)的水平下降约1/5,提示给MNU后一周内,光感受器的线粒体受到损伤,内侧其他视网膜细胞的线粒体尚存。给MNU后第3天,全视野ERG结果显示：最大混合反应的a波和b波波幅均明显下降。结论： MNU诱导模型主要导致光感受器细胞的丢失,并继发视网膜神经组织的结构重塑,其凋亡的机制可能是通过氧化损伤,该模型为研究RP和干性AMD等视网膜变性性疾病提供了有价值的动物模型。第二部分大麻素受体1抑制剂SR141716A缓解MNU诱导视网膜变性病变模型的光感受器损伤目的：研究大麻素受体1抑制剂SR141716A (SRI)缓解MNU诱导视网膜变性病变模型的光感受器损伤及机制。方法：造模组按50mg/kg腹腔注射给予N-亚硝基-N-甲基脲(MNU),对照组给予生理盐水和DMSO,干预组给于大麻素受体1和2的非特异性抑制剂WIN55,212-2(WIN)或SR1或SR144528(SR2).按照给药的药物成分,30只5周龄的C57/BL小鼠随机分为以下七组：MNU组、WIN组、SR1组、MNU+WIN组、MNU+SR1组、MNU+WIN+SRl组MNU+WIN+SR2组,观察给MNU后3天、5天、7天早中晚三个时期SR1对MNU造成的外核层损伤的影响。视网膜切片免疫荧光染色,显示大麻素受体1(CB1)在视网膜各层的分布。免疫荧光双标法显示CB1在视网膜各层细胞上的表达情况。急性分离细胞免疫荧光观察单个双极细胞上大麻素的表达。Real-time PCR定量观察大麻素受体mRNA水平的表达。免疫荧光染色观察给大麻素药物后光感受器厚度变化和胶质增生。全视网膜铺片免疫荧光染色血管显示血管损伤。薄片膜片钳技术记录ON型和OFF型双极细胞的对各种药物的电生理反应。结果：免疫荧光结果显示CB1在视网膜是有表达的,CB1在视网膜外丛状层和内丛状层的表达最为明显。用抗体OPN1SW、PKC a、Brn3a和GFAP分别标记光感受器细胞外节、双极细胞、神经节细胞的核和Muller细胞,分别与CB1抗体行免疫荧光双标,提示CB1在光感受器细胞外节、内核层双极细胞的胞体和神经节细胞的胞体也是有表达的,在Muller细胞上没有表达。急性分离的双极细胞用PKC α和CB抗体行免疫荧光双标,证实了双极细胞上的确有CB1的表达. Real-time PCR结果显示：MNU损伤后,大麻素受体1的基因表达量无变化,大麻素受体2基因表达上调。单独给WIN或WIN+SR1或WIN+SR2干预没有改善作用。单独给SR1干预有改善作用。与MNU组比较,MNU+SR1的外核层厚度更厚,胶质增生不明显。全视网膜铺片免疫荧光染色血管显示：MNU+SR1组的大血管损伤轻,只见少量的异常末梢血管丛。薄片膜片钳技术记录：ON型视杆细胞对MNU的损伤比OFF型双极细胞更敏感。SR1能部分缓解MNU诱导的光感受器损伤和及其对下游双极细胞的影响。结论：SR1能缓解MNU诱导视网膜外层变性模型的光感受器损伤。
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R774.1

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