【作者】 孙雅彬；

【导师】 宋鄂；

【作者基本信息】 吉林大学 ， 眼科学， 2013， 博士

【摘要】 目的：糖尿病性视网膜病变（diabetic retinopathy，DR）是全世界范围内工作人群致盲的首要原因[1]。眼内新生血管生成是DR病情发展的重要标志。血管内皮生长因子（vascular endothelial growth factor，VEGF）及其受体在血管新生、血管分化及增加血管通透性等方面起重要作用。视网膜Müller细胞在DR的发生发展过程起重要的作用。在高糖环境下Müller细胞产生VEGF增加。因此，基于视网膜Müller细胞在DR发病中的重要作用，本研究以体外培养的大鼠视网膜Müller增殖和蛋白表达变化入手，研究高糖培养的视网膜Müller细胞中VEGF蛋白质表达的调控机制。方法：原代培养大鼠视网膜Müller细胞，显微镜下观察细胞在体外培养过程中的形态变化、生长特性等，并且在细胞培养至第三代时收集并通过透射电镜观察。MTT法检测不同糖浓度中培养的视网膜Müller细胞的增殖情况。Müller细胞与生理（正常）葡萄糖浓度条件（5mM）和高糖条件（50mM）培养72h，ProteinPathway Array（PPA）法检测细胞中表达的磷酸化及非磷酸化蛋白质，应用146中磷酸化及磷酸化蛋白质的抗体进行检测，检测并分析出高糖条件下差异表达的蛋白质，为了更好的了解葡萄糖引起Müller细胞增殖的分子调控机制，我们应用Ingenuity PathwayAnalysis（IPA）软件对于表达调控蛋白质进行系统的分析。高糖培养的视网膜Müller细胞中，加入X连锁凋亡抑制蛋白（X-linked inhibitorof apoptosis protein，XIAP）的特异性抑制剂emblin培养72小时后，MTT法检测Müller细胞的增殖情况，利用PPA技术检测细胞内146种磷酸化和非磷酸化蛋白质的表达情况，并且通过IPA软件对差异表达蛋白之间的关系进行分析，构建信号传导通路。结果：高糖能够促进Müller细胞增殖，且增长呈葡萄糖浓度依赖性（50mM葡萄糖浓度是增殖最强），并且这种增殖作用能够被XIAP抑制剂embelin所抵消。高糖条件下Müller细胞内蛋白质表达发生显著变化，发现有108种磷酸化和非磷酸化蛋白质在正常糖浓度及高糖培养的视网膜Müller中表达，在视网膜Müller细胞中检测出47种磷酸化和非磷酸化蛋白质（如XIAP，VEGF，HIF1α，NF-κB等）表达具有显著性差异，这些蛋白质涉及细胞生存，增殖，凋亡等几个重要信号传导通路，其中VEGF和XIAP呈显著高表达，并且二者表达具有很强的相关性。XIAP的抑制剂embelin加入高糖培养环境后VEGF等26种蛋白质表达下降，同时Müller细胞中信号传导通路发生显著变化，embelin逆转了高糖所致的蛋白质表达变化。应用IPA软件分析并构建出，一条以XIAP为起点，有NFκB p65，p-p38，TNF-α，uPA，CREB，IL-1β，HCAM，Erα及p-Stat3等蛋白质参与，以VEGF为终点的信号通路。结论：高糖条件下的视网膜Müller细胞存在广泛的信号传导蛋白表达的异常；应用PPA方法检测高糖条件下异常调节信号蛋白是可行的。 VEGF蛋白质的表达受一条以XIAP为起点的信号通路所调控。本文研究提示XIAP可能参与调节高糖环境的视网膜Müller细胞的病理改变，XIAP可能通过NFkB复合物/p38通路调控VEGF的表达。因此，XIAP的特异性抑制剂emblin对DR有预防和治疗具有潜在作用。更多还原

【Abstract】 Purpose Müller cells play important roles in the pathogenesis of diabetic retinopathy(DR) by promoting cell proliferation and inducing the production of vascularendothelial growth factor (VEGF) under hyperglycemic conditions. The objective ofthis study was to determine the potential mechanism of Müller cell proliferation andVEGF production due to high glucose conditions.Methods Primary cultured rat Müller cells were incubated with medium containingvariable concentrations of glucose and/or embelin, a specific inhibitor of X-linkedinhibitor of apoptosis protein (XIAP), for72h. The proliferation of Müller cells wasassessed by the MTT assay. The expression and/or phosphorylation of146proteinswere assessed using Protein Pathway Array (PPA).Results We examined the effect of high glucose conditions on Müller cell growth usingthe MTT assay and found a dose-dependent increase of Müller cells after72h ofincubation with various concentrations of glucose (up to50mM) and the high glucoseinduced proliferation of Müller cells, which were reversed by embelin, a specificinhibitor of XIAP. High concentrations of glucose induced Müller cell proliferation andaltered expression and/or phosphorylation of47proteins that have been identified toplay key roles in several important signaling pathways (XIAP, VEGF, HIF1α, NF-κB,etc.) and are involved in the regulation of cell survival, proliferation, or apoptosis.However, Müller cell alterations induced by high glucose conditions werecounteracted by the XIAP inhibitor embelin, and26proteins/phosphorylations (out of47) were restored to their normal levels. Among146tested proteins andphosphoproteins,108were detected in protein samples from either the high or normalglucose treatments. Of which,47showed a significant difference in expression betweencells treated with high glucose. Forty proteins and phosphoproteins were overexpressedin high glucose-treated cells. These47proteins are involved in several importantsignaling pathways based on IPA, These data suggest that high glucose conditionscause a broad dysregulation of the signaling pathways in Müller cells. The PPA results showed that nine proteins, including NFκB p65, p-p38, TNF-α, uPA, CREB, IL-1β,HCAM, ERα, and p-Stat3, were involved in a regulatory network between XIAP andVEGF.Conclusions High glucose induced the significantly changes in the global network ofretinal Müller cells, which was successfully checked by PPA. The current studysuggests that XIAP may be a potential regulator that can mediate a series ofpathological changes induced by high glucose conditions in Müller cells. Wehypothesize that XIAP may regulate VEGF mainly through NFkB complex/p38cascade pathways. Therefore, embelin could be a potential agent for the prevention andtreatment of diabetic retinopathy.更多还原