摘要
糖尿病视网膜病变(diabetic retinopathy,DR)是糖尿病最为常见的慢性并发症,是当今世界上中老年龄段人群主要致盲性眼病之一。资料表明,糖尿病患者中,DR的发病率高达50%。15年病程的糖尿病患者,大约有10%因视网膜病变而有严重视力障碍,约2%成为盲目。
应用传统的研究方法,虽然已经初步揭示了DR的病理过程和部分相关机制,但是,缺乏对其复杂的生化和分子机制的全面了解。在临床上,对DR的预防和治疗,至今还没有理想的措施。蛋白质组学作为一门新兴学科,着重于从整体水平上研究生物体的功能。应用蛋白质组学的研究方法能够全面揭示组织或细胞内蛋白质的表达状况。通过比较正常和疾病状态下表达蛋白的差异,可揭示参与DR病理过程的多种因子和调控网络,发现新的与DR病理过程相关的蛋白。该技术对深入、系统的研究DR发生发展的病理机制,确定有效预防和治疗药靶方面有独特的优势。
本研究第一部分,对SD大鼠神经视网膜进行分离,并用组织学和透射电镜方法鉴定所分离的组织,提取组织总蛋白,应用二维电泳(two-dimensional gel electrophoresis,2DE)技术对提取的蛋白进行分离,摸索各个实验环节的条件,获得了分辨率高和重复性好的2DE图像,建立了2DE分离技术的平台,为进一步研究奠定了基础。
本研究的第二部分,用高脂饮食联合小剂量STZ(streptozotocin,STZ)腹腔注射建立大鼠2型糖尿病模型。以年龄匹配正常SD大鼠为对照。分离神经视网膜组织,提取组织总蛋白。2DE分离蛋白质,应用银染或考马斯兰染色方法显示分离的蛋白质。通过计算机图像分析系统对凝胶图像进行处理,在24cm,pH3-10的胶体上,正常组平均每块胶检测到3122±37(n=3)个蛋白点;糖尿病组,平均每块胶检测到2702±21(n=3)个蛋白点,平均匹配率为76.8%。大部分蛋白质点分布在pI 4-8和分子量14.4-64.4KD区域。与正常组比较,表达有明显差异的约150个蛋白点。在糖尿病组表达上调的点68个,下调的点82个。
采用肽质量指纹谱(peptide mass fingerprinting,PMF)和串联质谱肽序列分析方法对差异表达蛋白进行鉴定。经鉴定,得分值较高的20个蛋白质中,有10个为新发现的蛋白,值得进一步研究。初步分析表明,差异蛋白质的功能涉及糖代谢和脂代谢、炎症反应、NO合酶途径以及细胞骨架蛋白和分子伴侣蛋白等。
本研究第三部分,应用western blot方法和间接免疫荧光(immunofluoresce,IMF)方法显示糖尿病大鼠神经视网膜组织αA-晶体蛋白表达上调。IMF显示αA-晶体蛋白主要分布在视网膜核层。验证了2DE的结果。
本研究第四部分,应用组织学方法显示早期糖尿病大鼠视网膜神经节细胞密度降低。视网膜消化铺片方法没有发现微血管周细胞和内皮细胞有明显异常变化。提供了糖尿病早期视网膜神经细胞退行性病变的依据。
本研究应用蛋白质组学的技术和方法,分离和鉴定了早期糖尿病大鼠神经视网膜差异表达蛋白。发现了一些新蛋白。用western blot和间接免疫荧光法验证2DE结果。组织学方法和视网膜消化铺片结果证明糖尿病早期视网膜神经组织退行性病变早于微血管病变。本研究实验方法先进,技术稳定,结果可靠,提供了DR发病机制的相关依据。
Diabetic retinopathy(DR), a common chronic complication ofdiabetes, is one of leading causes of blindness and visual disability inelderly worldwide. The data indicate that the prevalence rate of DR isabout 50.0% in diabetes. Approximately 10% of patients with diabetes formore than 15 years have developed severe visual impairment, and about2% have become blind.
Although the pathological process and correlative pathologicmechanism of DR have been widely studied by using conventionalmethods, the biochemical and molecular pathways that underlie theinitiation and progression of diabetic retinopathy remain unresolved andthere is no effective means to prevent and cure DR even today.Proteomics, a new approach to study of diseases, is not to study ofindividual protein as traditionally, but rather to study the entirecomplement of proteins expressed by a cell or tissue at a given time.The multiple factors and the complex regulatory network that are responsible for pathological process of DR can be revealed by comparingthe protein expression profiles from normal tissue to those frompathological tissue, and also the cellular and molecular mechanism of DRbe unraveled.
In the first section of the present study, neural retinas were dissectedfrom eyes of Sprague-Dawley (SD) rats and separated neural retinas wereidentified by histopathology and transmission electron microscope (TEM).Total proteins of neural retina were extracted and high-resolution 2DEwas used to separate total proteins. The experiments were repeated tooptimize the experiment conditions of the isoelectric focusing (IEF),SDS polyacrylamide gel electrophoresis(SDS-PAGE), silver staining(or coomassie-blue-staining) and 2DE techniques. Well resolved,reproducible 2DE profiles of rat neural retinas were establised. Theresults provide a basis for further research on the diabeticretinopathy.
In section two, normal age-matched control SD rats and 8 weeksdiabetic rats induced by feeding a high-fat(HF) diet combined withintraperitoneal(i.p.) injection of streptozotocin (STZ) were researched.Total proteins were extracted from neural retinas of two groups of rats,then high-resolution 2DE was used to separate total proteins followed bysilver staining or coomassie-blue-staining. Computer assisted imageanalysis(Image Master 5.0) to quantitative compare and analyze the differences in protein expression between age-matched control rats anddiabetic rats. Total 3122±37(n=3) spots were detected in normal samplesand 2702±21(n=3) spots in diabetic rats. The average matching rate was76.8%. Most protein spots were visualized within a pI range of 4.0-8.0and a molecular mass range from 14.4 to 66.4 kDa. It was found byimage analysis that about 150 spots in 2DE gel of diabetic retinas thatexhibited statistically significant variations, including up-regulation of 68proteins in diabetic rat retinas, down-regulation of 82 compared withnormal rat retinas. Twenty differentially expressed proteins wereidentified by peptide mass fingerprinting (PMF) or peptide sequence, inwhich ten proteins were newly discovered proteins associated with DR.The functions of identified proteins were primary analyzed. Thedifferentially expressed proteins revealed by our experiments werediverse, including proteins involved in saccharide and lipid metabolism,inflammatory response, nitric oxide synthetase pathway, as well ascytoskeletal and molecular chaperone proteins.
Then, the proteinαA-crystallin was chosen as a target for specificimmunodetection using western blot. Moreover, immunofluorescence(IMF) staining was carried out to demonstrate the expression and locationofαA-crystallin in the retina. The resultes corroborate the variation foundby 2DE.
A histological method(HE staining)was used to detect the neuronal degeneration of diabetic retina and found the decreased density ofganglion cell in the retinas from diabetes group.
In short, using a proteomics approach we separated and identifiedproteins differentially expressed in the diabetic retinas and find somespots were probably novel proteins. Western blot and immunofluore-scence staining were used to corroborate the variation found by 2DE. Ahistologic method demonstrated neuronal degeneration of diabetic retinain the early stage of diabetes. The methods applied in present study werestable and the results were reliable. All data provided a new understand tothe pathomechanism of the DR.
引文
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