【作者】 韩世杰；

【导师】 邱贵兴；

【作者基本信息】 北京协和医学院 ， 骨外科学， 2011， 博士

【摘要】 研究背景：退行性脊柱侧凸(degenerative scoliosis, DS)随着人口老龄化及老年人生活方式的转变,已成为导致脊柱畸形、功能障碍、影响生活质量的一种重要的腰椎退行性疾病。DS的病因尚不明确,可能与椎间盘、关节突关节的不对称退变、骨质疏松和遗传学因素有关,但均没有明确证据可以解释其病因。目前关于该病的研究绝大多数都是针对该病的治疗,涉及病因学方面的基础研究很少,而且还没有分子生物学方面的报道。因此,弄清DS的病因及发病机制具有重要意义。骨髓间充质干细胞(bone merrow-derived mesenchymal stem cell,BM-MSC)是成骨细胞的干/祖细胞,可以在体内不断分化为骨组织的各种成分,成骨相关的疾病很可能是在干细胞水平上发生了异常而产生疾病,因此从MSC水平对骨病成因进行研究很有必要。蛋白质组学是一项新兴的实验技术,能从蛋白质的整体水平,更深入、更接近生命本质的层次去发现和探讨生命活动的规律及其重要生理病理现象的本质。而MSC的蛋白质组学研究刚刚起步,尤其是对于疾病的比较蛋白质组学研究更是少有报道。2D-DIGE是近年来发展成熟的蛋白质组技术,是目前定量蛋白质组学研究中可信度和准确性最高的技术之一。本研究旨在首先培养和鉴定DS患者和对照组(腰椎管狭窄患者)的MSC,然后采用蛋白质组技术从MSC的水平寻找DS与对照组的差异蛋白,并对DS的发病机制和病因进行探讨,为进一步从基因层面研究提供依据。研究目的：1.分离和培养扩增DS患者和对照组的MSC,并对其进行鉴定。2.构建DS患者与对照间MSC差异蛋白质组图谱。3.应用质谱鉴定技术,对差异蛋白质点进行鉴定。4.结合蛋白功能网络研究成果,分析所鉴定的蛋白质在DS发生发展中的作用,并为在基因层面的进一步研究提供依据。研究方法：1.采集12例退行性脊柱侧凸患者和12例年龄、性别匹配的腰椎管狭窄患者的骨髓,分离、培养扩增MSC,制备MSC蛋白质样品。并采用流式细胞仪检测细胞表面抗原,成骨诱导体系及脂肪诱导体系分别诱导MSC定向分化,通过细胞形态观察及免疫化学染色进行鉴定。2. CyDye染料交叉标记,然后进行荧光双向差异凝胶电泳、图像扫描,并用DeCyderv.5.02图像分析软件对DIGE图像进行分析和寻找差异点。3.对筛选出的差异点进行基质辅助激光解吸／电离飞行时间质谱(MALDI-TOF-MS)分析,检索IPI-HUMAN数据库鉴定蛋白。研究结果：1.从DS和腰椎管狭窄患者的骨髓液中分离出MSC。这些MSC稳定表达CD29、CD44、CD105,不表达CD31、CD34、CD45。定向诱导的成骨细胞表达碱性磷酸酶活性,脂肪细胞内出现明显的脂滴。2.经过荧光双向差异凝胶电泳、DeCyder软件分析发现,DS患者和腰椎管狭窄患者MSC中共有115个蛋白质差异点,两者之间的差异有统计学意义。其中有70个点在DS干细胞中上调,有45个点表达下调。3.115个蛋白质差异点经质谱分析,IPI-HUMAN数据库鉴定44个差异蛋白。结论：1.应用密度梯度离心联合贴壁筛选法并通过流式细胞术检测及定向诱导分化,能够从DS和对照组患者的骨髓液中分离出MSC,并完成MSC表型和多向分化的初步鉴定。2.首次对DS患者进行MSC水平的蛋白质组研究,构建了MSC差异蛋白质组资料库。3.应用质谱鉴定技术,成功鉴定出44个差异表达蛋白质。这种蛋白质的差异表达可能更接近DS发生的使动因素。其具体意义、具体作用及其是否具有DS特异性尚需进一步研究以阐明。研究结果为DS的发病和进展机制研究奠定了理论基础,为在基因层面的进一步研究提供依据。更多还原

【Abstract】 Background:As the population aging trends going on and the lifestyle changing of the elderly, Degenerative scoliosis (DS) has become more and more important in the degenerative lumbar disease causing spinal deformity, dysfunction and affecting quality of life of the elderly. Although osteoporosis, asymmetric degenerative disc disease, facet tropism and inheritance have been implicated as factors in the development of DS, none of these has been shown to be directly related. The pathogenesis of DS is still unknown. Most current researches on the DS are focused on the therapeusis, but the basic research on the etiology is few. Furthermore, there has no molecular biology study been reported. So it is of great importance to find out the true reason or pathogenesis of DS.Bone merrow-derived mesenchymal stem cells(BM-MSCs) are the stem/progenitor cells of the osteoblasts. They can differentiate into several components of the bone, and the diseases associated with osteogenesis could be caused by the abnormity of the MSCs. So we should investigate the bone disease on the level of MSCs.Proteomics is a newly emerging experiment technology, it can find the differences on the level of the whole proteins to investigate the rule or nature of several vital movements and physiology or pathology phenomenons. The proteomics researches on the MSCs are just starting and rare, particularly the comparative proteomics researches on the disease. The recently introduced differential in gel electrophoresis(DIGE) technology is believed to be a highly reliable and accurate tool for quantitative analysis of differentially expressed proteins.In our study, MSCs of patients of DS and the Control(lumbar spinal stenosis) were isolated, cultured and identified. Then, the Comparative Proteomics Research on MSCs was performed to find the differential proteins which was studied and discussed to provide effective biochemical evidence for the coming gene researches and help to undersand the pathogenesis of DS.Objectives:1. To isolate and culture the MSCs of the patients of DS and the Control, and to identify the MSCs. 2. To establish two dimensional difference in gel electrophoresis(2D-DIGE) of MSCs of DS and the control.3. To identify the differential proteins expressed in MSCs of patients using mass spectrometry techniques.4. To analyze the role of the identified proteins in the development of degenerative scoliosis through the present protein functional network of research results.Methods:1. Bone marrow was collected from 12 degenerative scoliosis patients and 12 age-and gender-matched patients with lumbar spinal stenosis. Then the MSCs was harvested after being differentiated, cultured and identified.2. The samples were labeled with different CyDye, followed by 2D-DIGE, map scanning, and then the maps of protein spots were compared using specific software DeCyder v5.02.3. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and IPI-HUMAN data base.Results:l.By gradient centrifugation and adhesion culture, MSCs could be isolated and culture-expanded from human bone marrow aspirate. These cells were uniformly negative for CD31、CD34、CD45 and positive for CD29、CD44、CD105. The osteogenic differentiated cells were positive for alkaline phosphatase(ALP) and the adipogenic differentiated cells displayed accumulation of lipid vacuoles as detected by oil red O.2.115 spots that were differently expressed in the MSC of DS patients were found and identified. There were 70 proteins significantly up-regulated and 45 spots significantly down-regulated in MSC of DS patients.3. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).44 proteins were identified from samples of DS and Control.Conclusions:1. MSCs can be isolated and expand-cultured from adult human bone marrow aspirate and committedly differentiate into osteoblasts and adipocytes. MSCs primary identification can be accomplished by flow cytometry and induced differentiation.2. This is the first time to study comparative proteomics on MSC of DS. And the MSC 2D-DIGE of DS patients and lumbar spinal stenosis were successfully settled.3. There were 44 protein spots of significance between the MSC of patients with DS and lumbar spinal stenosis analyzed by mass spectrometry techniques. Though the result is preliminary, but this differences of the protein may be the original factors of DS. Further investigations are necessary to illustrate the functioning pathway, the specificity and the mechanism of how these proteins contribute to pathogenesis of DS. The information obtained with this proteomic analysis will be very useful in understanding the pathophysiology of DS and finding the pathogenic genes of DS.更多还原