卵巢癌及癌旁正常卵巢组织中USP2、USP14和UBE4A的差异表达

目的:探讨泛素特异性蛋白酶(USP)2、14和泛素因子E4A(UBE4A)在卵巢浆液性囊腺癌及癌旁正常卵巢组织间的表达差异。方法:采用RFDD-PCR、半定量RT-PCR和免疫组织化学染色方法检测USP2、USP14和UBE4A在40例卵巢浆液性囊腺癌患者卵巢癌组织及其癌旁正常卵巢组织上的表达差异。结果:泛素特异性蛋白酶USP2、USP14和UBE4A在卵巢浆液性囊腺癌组织中高表达,而在正常卵巢组织中无表达或低表达。结论:USP2、USP14和UBE4A在卵巢癌组织中表达上调,提示卵巢癌中泛素-蛋白酶体系统活动明显增强。     

糖尿病大鼠视网膜基因表达谱差异的初步分析

目的 建立大鼠正常视网膜和糖尿病8周视网膜基因表达谱，比较两者差异，初步分析糖尿病视网膜病变的相关基因。方法 通过限制片段差异显示 PCR（ restriction fragments differential display-PCR，RFDD-PCR）获得正常大鼠视网膜及8周糖尿病大鼠视网膜组织转录组片段。应用Fraent Analysis等软件，对差异片段进行生物信息学分析，初步确定糖尿病视网膜病变相关基因／表达序列标签（ expression sequence tag, Ksr）。结果 获得有意义的片段共3639个，有差异的片段840个，占表达数的23．08％。其中包括5个视觉传导相关基因，13个兴奋性神经递质受体基因和3个抑制性神经递质受体基因。糖尿病8周大鼠视网膜Rhodopsin kinase,β-arrestin,Phosducin, rod photoreceptor cGMP-gated channel 和 Rpe65的表达下调，离子型谷氨酸受体iGluR1-4下调，代谢性谷氨酸受体及γ-氨基丁酸受体各亚型则普遍上调，而甘氨酸受体表达无变化。结论 糖尿病8周大鼠神经视网膜已受到累及，其基因表达模式的改变，可能与糖尿病早期视功能损害有关。     

大鼠视神经切断后视网膜双极细胞PKC-α和recoverin的表达

为了探讨视神经切断后视网膜内部是否存在突触可塑性改变,本实验采用大鼠视神经切断模型,通过免疫组织化学方法检测视神经切断后视网膜双极细胞PKC-α和recoverin的表达变化。结果显示:正常视网膜中,PKC-α和recoverin阳性产物主要见于视网膜内核层、内网层及节细胞层,另外外核层也可见少量recoverin阳性细胞。视神经切断后3d,大鼠视网膜内网层高倍镜下可见PKC-α和recoverin免疫阳性终末的数量开始增加,14d时增至最高,21d、28d呈现逐渐减少的趋势。本研究结果提示视神经切断后视网膜双极细胞与节细胞之间的突触可能存在早期增生,后期溃变的可塑性变化。     

大鼠,金黄地鼠和家兔视网膜内一氧化氮合酶分布的比较

用ＮＡＤＰＨ黄递酶组织化学染色法观察了正常成年大鼠、金黄地鼠和家兔视网膜内一氧化氮合酶的分布，并比较了３种不同动物的区别。结果显示，在视网膜内ＮＯＳ阳性神经元主要为分布于内核层的无长突细胞、节细胞层的移位无长突细胞和少数节细胞，不同种类动物的视网膜内，ＮＯＳ阳性细胞的配布、密度和细胞形态均有差异。大鼠视网膜内ＮＯＳ阳性细胞多尾于内核层无长突细胞和节细胞层移位无长细胞，偶见于视网膜节细胞。金黄地鼠视     

猫视网膜S100蛋白和髓鞘碱性蛋白表达的老年性变化

目的:比较老年猫与青年猫视网膜S100与髓鞘碱性蛋白(MBP)表达的变化.方法:用免疫组织化学ABC法显示S100、MBP阳性结构.显微镜下观察S100、MBP阳性反应,并对S100阳性细胞进行计数.结果:老年猫、青年猫S100免疫反应阳性结构均见于神经节细胞层和神经纤维层的星形胶质细胞.与青年猫相比较,老年猫视网膜中S100阳性反应强于青年猫,阳性细胞显著增加.老年猫MBP免疫反应阳性结构见于外网状层和内网状层的神经纤维,青年猫阳性反应很弱.结论:猫视网膜衰老过程中S100和MBP表达增强.     

实验性早期糖尿病大鼠视网膜水通道蛋白4及其mRNA的表达变化

目的:探讨实验性早期糖尿病鼠视网膜水通道蛋白4(AQP4)及其mRNA的表达变化及其意义.方法:用链脲菌素(streptozotocin,STZ)腹腔注射的方法建立大鼠糖尿病模型,运用墨汁灌注实验检测视网膜微血管渗漏情况;用免疫荧光组织化学检测AQP4存视网膜内的分布和表达变化;用原位杂交和RT-PCR检测AQP4 mRNA在视网膜内的分布及表达变化.结果:与同龄正常对照组大鼠相比,早期糖尿病鼠视网膜末见明显血管渗漏现象,但存在部分细胞水肿、节细胞数目减少、感光细胞外节排列紊乱等病理改变;免疫组织化学、原位朵交和RT-PCR结果显示.模型鼠视网膜内AQP4及其mR-NA的表达明显增强.结论:实验性早期糖尿病鼠视网膜.在微血管病变发生前,已存存细胞水肿等病理改变;AQP4及其mRNA的表达明显上调.AQP4表达上调可能是糖尿病早期视网膜水肿形成、视功能下降的重要原因.     

MCP-1及其受体在wAMD小鼠模型视网膜中的表达及作用机制

目的探讨单核细胞趋化因子-1(monocyte chemoattractant protein-1,MCP-1)及其受体CCR2在湿性年龄相关性黄斑变性(wet age-related macular degeneration,wAMD)小鼠模型视网膜中的表达及作用机制。方法制备wAMD小鼠模型,摘取眼球制作冷冻切片,使用MCP-1和CCR2单克隆抗体行免疫荧光检测。使用CD11b和CD68单克隆抗体分别与CCR2抗体进行免疫荧光共表达研究以检测CCR2阳性细胞的来源。制备视网膜的总蛋白和总mRNA提取物,经RT-PCR和Western blot检测视网膜中MCP-1及其受体CCR2 mRNA和蛋白的表达。结果正常对照组小鼠视网膜中无明显的MCP-1表达,wAMD小鼠模型中视网膜色素上皮(retinal pigment epithelium,RPE)层下有显著的新生血管网形成,且RPE层连续性被破坏,其中RPE细胞有明显的MCP-1表达上调。正常对照组小鼠视网膜中无明显的CCR2的表达,wAMD模型小鼠的视网膜中CCR2阳性细胞显著增加。与正常对照组小鼠相比,wAMD模型小鼠眼内MCP-1和CCR2的蛋白和mRNA表达明显上调。免疫荧光共表达结果显示CCR2与CD11b有显著的共表达,双阳性细胞集中在RPE层和视网膜外层;而CCR2与CD68无明显的共表达。结论 MCP-1及其受体可能与wAMD小鼠模型视网膜脉络膜新生血管(choroidal neovascularization,CNV)的形成及其炎症机制相关。     

基于公共数据库挖掘UBE2A在肝细胞癌中的表达与调控及其临床意义

目的:探讨泛素结合酶 2A(Ubiquitin binding enzyme 2A,UBE2A)在肝细胞癌(Hepatocellular carcinoma,HCC)中的表达与调控因子、预后及其与免疫细胞浸润的关系.方法:通过Timer2.0 数据库初步分析UBE2A在不同肿瘤中肿瘤和正常组织的表达差异;进一步使用GEPIA数据库分析肝细胞癌中UBE2A的表达情况及其与预后的关系.在 GEO 数据库中筛选肝细胞癌数据集(GSE108724)中差异表达 miRNA,并利用 mirDIP 工具构建gene-miRNA调控网络.使用UALCAN数据库探索UBE2A与临床病理特征的关系.Timer数据库分析出肝细胞癌中UBE2A与免疫细胞浸润的关系.结果:肝细胞癌组织中UBE2A基因表达高于正常组织(P＜0.05),UBE2A高表达患者预后较差(P=0.045).hsa-miR-106b-5p、hsa-miR-375、hsa-miR-93-5p在肝细胞癌中参与UBE2A的调控.UBE2A的表达与年龄、性别、淋巴结转移无关(P＞0.05),与肿瘤分期相关(P＜0.05).UBE2A的表达与肝细胞癌中B细胞、CD8+ T细胞、CD4+ T细胞、巨噬细胞、中性粒细胞和树突状细胞的浸润水平呈正相关.结论:UBE2A有望成为肝细胞癌诊断的新型标志物和治疗的潜在分子靶点.     

丝胶预处理对2型糖尿病大鼠腺垂体内NPY及leptin表达的影响

目的:观察彩色蚕茧提取物-丝胶对2型糖尿病大鼠腺垂体内神经肽Y(NPY)及leptin表达的影响。方法:将36只SD大鼠随机分为正常组、模型组和丝胶预处理组。模型组、丝胶预处理组大鼠均建立链脲佐菌素(STZ)致2型糖尿病动物模型,丝胶预处理组于注射STZ前给予丝胶灌胃35 d。采用酶法检测各组大鼠血糖、免疫组织化学染色和RT-PCR观察各组大鼠腺垂体内NPY和leptin蛋白及mRNA的表达。结果:血糖监测结果显示丝胶预处理组大鼠血糖明显低于模型组(P0.01);免疫组织化学染色结果显示:NPY和leptin免疫阳性产物均为棕黄色颗粒状,免疫阳性细胞均为腺垂体内嗜色细胞,NPY主要位于细胞质,leptin主要位于细胞核。与模型组大鼠相比,丝胶预处理组大鼠NPY和leptin蛋白的表达明显降低(P0.01);RT-PCR结果显示:丝胶预处理组大鼠NPY和leptin mRNA的表达明显低于模型组(P0.01)。结论:丝胶预处理可下调腺垂体内NPY和leptin的高表达,对2型糖尿病腺垂体损伤具有明显的预防作用。     

糖尿病视网膜病变患者检测糖化血红蛋白的临床意义

目的探讨糖尿病视网膜病变 (DR)患者检测糖化血红蛋白 (HbA1c)的临床意义。方法对154例2型糖尿病患者DR情况和HbA1c水平进行检测。并对71例、病程5～10年的糖尿病患者正常眼底 (NDR)或单纯型糖尿病视网膜病变 (BDR)患者进行1年的随访 ,检测DR进展情况和HbA1c水平。结果DR组HbA1c高于NDR组。增殖型糖尿病视网膜病变 (PDR)组HbA1c高于BDR组。随访1年 ,视网膜病变进展组(A组)平均HbA1c高于视网膜病变稳定组(B组),差异具有统计学意义 (P<0.01)。结论DR的程度和进展与HbA1c增高有关     

Expression of Glutamate and GABA during the Process of Rat Retinal Synaptic Plasticity Induced by Acute High Intraocular Pressure

Acute high intraocular pressure (HIOP) can induce plastic changes of retinal synapses during which the expression of the presynaptic marker synaptophysin (SYN) has a distinct spatiotemporal pattern from the inner plexiform layer to the outer plexiform layer. We identified the types of neurotransmitters in the retina that participated in this process and determined the response of these neurotransmitters to HIOP induction. The model of acute HIOP was established by injecting normal saline into the anterior chamber of the rat eye. We found that the number of glutamate-positive cells increased successively from the inner part to the outer part of the retina (from the ganglion cell layer to the inner nuclear layer to the outer nuclear layer) after HIOP, which was similar to the spatiotemporal pattern of SYN expression (internally to externally) following HIOP. However, the distribution and intensity of GABA immunoreactivity in the retina did not change significantly at different survival time post injury and had no direct correlation with SYN expression. Our results suggested that the excitatory neurotransmitter glutamate might participate in the plastic process of retinal synapses following acute HIOP, but no evidence was found for the role of the inhibitory neurotransmitter GABA.     

Expression of the 40 kDa catecholamine regulated protein in the normal and injured rat retina

Catecholamine regulated protein 40 (CRP40) has been shown to be expressed in the central nervous system (CNS) of several mammalian species where it may function in a similar manner to members of the heat shock protein (HSP) family. Immunohistochemical and immunoblotting techniques were utilized to investigate whether CRP40 is expressed in normal rat retinas. In addition, changes in CRP40 expression were studied following optic nerve transection. The immunohistochemical results showed that CRP40 is expressed in the normal rat retina. The protein was found to be highly expressed in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer plexiform layer (OPL). In addition, a low level of CRP40 was found in the inner plexiform layer (IPL), and in the inner segment layer (ISL). No expression was found in the outer nuclear layer (ONL) of normal rat retina. The immunoblotting results show that CRP40 expression decreased in a time-dependent fashion after the optic nerve transection. This decrease indicates that the expression of CRP40 is dependent on the neuron's normal physiological state and that it plays an important function in physiological and pathological conditions in the retina.     

Dopaminergic transmission in the rat retina: evidence for volume transmission

The study was designed to determine whether dopaminergic neurotransmission in the retina can operate via volume transmission. In double immunolabelling experiments, a mismatch as well as a match was demonstrated in the rat retina between tyrosine hydroxylase (TH) and dopamine (DA) immunoreactive (ir) terminals and cell bodies and dopamine D2 receptor-like ir cell bodies and processes. The match regions were located in the inner nuclear and plexiform layers (D2 ir cell bodies plus processes). The mismatch regions were located in the ganglion cell layer, the outer plexiform layer, and the outer segment of the photoreceptor layer, where very few TH ir terminals can be found in relation to the D2 like ir processes. In similar experiments analyzing D1 receptor like ir processes versus TH ir nerve terminals, mainly a mismatch in their distribution could be demonstrated, with the D1 like ir processes present in the outer plexiform layer and the outer segment where a mismatch in D2 like receptors also exists. The demonstration of a mismatch between the localization of the TH terminal plexus and the dopamine D2 and D1 receptor subtypes in the outer plexiform layer, the outer segment and the ganglion cell layer (only D2 immunoreactivity (IR)) suggests that dopamine, mainly from the inner plexiform layer, may reach the D2 and D1 mismatch receptors via diffusion in the extracellular space. After injecting dopamine into the corpus vitreum, dopamine diffuses through the retina, and strong catecholamine (CA) fluorescence appears in the entire inner plexiform layer and the entire outer plexiform layer, representing the match and mismatch DA receptor areas, respectively. The DA is probably bound to D1 and D2 receptors in both plexiform layers, since the DA receptor antagonist chlorpromazine fully blocks the appearance of the DA fluorescence, while only a partial blockade is found after haloperidol treatment which mainly blocks D2 receptors. These results indicate that the amacrine and/or interplexiform DA cells, with sparse branches in the outer plexiform layer, can operate via volume transmission in the rat retina to influence the outer plexiform layer and the outer segment, as well as other layers of the rat retina such as the ganglion cell layer.     

Expression and cell localization of brain-derived neurotrophic factor and TrkB during zebrafish retinal development

Brain‐derived neurotrophic factor (BDNF) signaling through TrkB regulates different aspects of neuronal development, including survival, axonal and dendritic growth, and synapse formation. Despite recent advances in our understanding of the functional significance of BDNF and TrkB in the retina, the cell types in the retina that express BDNF and TrkB, and the variations in their levels of expression during development, remain poorly defined. The goal of the present study is to determine the age‐dependent changes in the levels of expression and localization of BDNF and TrkB in the zebrafish retina. Zebrafish retinas from 10 days post‐fertilization (dpf) to 180 dpf were used to perform PCR, Western blot and immunohistochemistry. Both BDNF and TrkB mRNAs, and BDNF and full‐length TrkB proteins were detected at all ages sampled. The localization of these proteins in the retina was very similar at all time points studied. BDNF immunoreactivity was found in the outer nuclear layer, the outer plexiform layer and the inner plexiform layer, whereas TrkB immunoreactivity was observed in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer. These results demonstrate that the pattern of expression of BDNF and TrkB in the retina of zebrafish remains unchanged during postembryonic development and adult life. Because TrkB expression in retina did not change with age, cells expressing TrkB may potentially be able to respond during the entire lifespan of zebrafish to BDNF either exogenously administered or endogenously produced, acting through paracrine mechanisms.     

Neuronal expression of P2X3 purinoceptors in the rat retina

P2X3 purinoceptors are involved in fast, excitatory neurotransmission in the nervous system, and are expressed predominantly within sensory neurons. In this study, we examined the cellular and synaptic localization of the P2X3 receptor subunit in the retina of the rat using immunofluorescence immunohistochemistry and pre-embedding immunoelectron microscopy. In addition, we investigated the activity of ecto-ATPases in the inner retina using an enzyme cytochemical method. The P2X3 receptor subunit was expressed in the soma of a subset of GABA immunoreactive amacrine cells, some of which also expressed protein kinase C-alpha. In addition, punctate immunoreactivity was observed within both the inner and outer plexiform layers of the retina. Double labeling studies showed that P2X3 receptor puncta were associated with both rod and cone bipolar cell axon terminals in the inner plexiform layer. Ultrastructural studies indicated that P2X3 receptor subunits were expressed on putative A17 amacrine cells at sites of reciprocal synaptic input to the rod bipolar cell axon terminal. Moreover, we observed P2X3 immunolabeling on amacrine cell processes that were associated with cone bipolar cell axon terminals and other conventional synapses. In the outer retina, P2X3 immunoreactivity was observed on specialized junctions made by putative interplexiform cells. Ecto-ATPase activity was localized to the inner plexiform layer on the extracellular side of all plasma membranes, but was not apparent in the ganglion cell layer or the inner nuclear layer, suggesting that ATP dephosphorylation occurs exclusively in synaptic regions of the inner retina. These data provide further evidence that purines participate in retinal transmission, particularly within the rod pathway.