【作者】 尹若峰；

【导师】 邱贵兴； 王以朋； 翁习生；

【作者基本信息】 中国协和医科大学 ， 外科学， 2009， 博士

【摘要】 研究背景:青少年特发性脊柱侧凸(AIS)是一种脊柱三维畸形,当青少年脊出现结构性侧凸(冠状面X线平片上Cobb角>10°,合并脊柱的旋转畸形),而无其他器质性疾病时,可以临床上诊断此疾病。AIS是儿童及青少年骨骼肌肉系统最常见的畸形之一。正如它命名中“特发性”所指,尽管学者们做了大量研究,但迄今为止它的病因仍然不明,这就给它的早期干预、早期治疗带来了一定困难。尽管AIS的发病原因尚不清楚,但经过多年的研究,一些科学家或科研机构一经提出了可能与脊柱侧凸发生和发展相关的一些假说:如家族遗传因素学说,生长发育学说和结缔组织异常学说等诸多观点问世,但各种论点所得出的结论都不能很好地进行重复性试验。所以大部分学者认为,AIS是一个涉及多遗传易感基因位点和多系统致病环节,引发多种损害效应的蛋白质分子表达异常的疾病。单基因和单一蛋白质分子的研究已经进入了一条无法继续前行的死胡同。从脊柱侧凸病因学的报告文献中可以发现,近五年来已经没有新的观点或发现问世。椎间盘不对称是引起AIS畸形产生和进展的结构基础。从解剖学和影像学检查中不难发现,凸侧椎问盘的发育较凹侧明显成熟。从解剖学和影像学检查中不难发现,凸侧椎间盘的发育较凹侧明显成熟。椎间盘由纤维环环绕凝胶状的髓核及覆盖于此结构的上下软骨终板组成。软骨终板通常厚度不到1mm,由透明软骨组成,它将椎体和间盘纤维环及髓核隔开。。椎间盘作为人体内最大的无血管组织,除纤维环外围极少数几层环状纤维板通过临近血管进行营养代谢外,其余椎间盘组织则需要通过软骨终板的弥散作用完成营养获取和代谢物的排出。Driscoll M等研究发现椎间盘的不均衡发育程度和AIS的进展呈正相关;另外,与Lonstein JE的结论相同,并且通过比较凸、凹侧椎间盘差异的大小可以预测AIS患者侧凸畸形的进展速度和畸形所能达到的严重程度。研究发现,凸、凹侧脊柱软骨终板的细胞活动和组织成熟度差异有可能影响脊柱侧凸的进展。组织学研究表明,侧弯顶椎凸、凹侧软骨终板的细胞活动和组织成熟度存在明显的差异。在脊柱后凸畸形和侧凸畸形的患者中,能够观察到椎间盘出现形明显的态学上变化。其中最为引人注目的一点就是在脊柱侧凸患者的软骨终板内不同程度异常钙化的出现。这种钙化的通常由细胞的两极开始出现,逐渐环绕整个细胞,进而扩散至到细胞外基质。这种异常钙化影响了椎间盘组织的营养供应和物质代谢,进而可能影响椎间盘的生长和发育。AIS脊柱畸形通常开始于青春期患者生长发育起始阶段,而此前并未出现脊柱的侧凸畸形及椎间盘的不对称生长。在由正常-异常的生长发育过程中,侧凸部位的椎间盘和终板的必然发生了某些变化。蛋白质作为生物生理功能过程中最重要的一类分子,必然会在这种变化中发挥重要的作用。近年来蛋白质组学研究技术的发展为AIS的研究带来了新的动力。通过对比不同组织样品中的蛋白质组,我们可以直观地了解二者之间在分子水平上有何不同。研究目的:1.分别鉴定凸侧和凹侧椎间盘软骨终板的蛋白质组2.比较二者之间的差异3.尝试寻找引起椎间盘不对称发育的关键蛋白研究方法:1.研究对象:根据纳入和排除标准确定2006年7月-2007年8月期间北京协和医院脊柱外科收治并确诊的6例青少年脊柱侧凸(AIS)患者。2.技术方法:(?)侧凸顶点软骨终板组织获取和处理:通过前路脊柱侧凸矫形手术,手术医师在术中完整取得侧凸顶点软骨终板;去除软骨组织表面附着的纤维环和骨组织,充分清洗确保无血细胞残留。(?)软骨组织蛋白质提取:使用组织切片、洗脱、蛋白质沉淀技术直接获取软骨组织蛋白质。(?)测定蛋白质浓度并进行SDS-PAGE蛋白质样品电泳分析。(?)质谱分析:使用二维液相色谱串联电喷雾离子阱质谱(2D-LC-LTQ)技术进行蛋白质质谱分析(?)数据处理:质谱数据使用SEQUEST对human ref蛋白质数据库(Version 9.18)进行解读,并用Bioworks3.2将结果进行过滤,分类和显示。由SwissProt和TrEMBL蛋白库的搜索结果给蛋白标注上名称。研究结果:(?)软骨组织中获得的平均蛋白质浓度为3.02mg/ml(2.20-3.97 mg/ml)。(?)聚丙烯酰胺凝胶蛋白电泳(SDS-PAGE)发现,凸侧蛋白质样品在蛋白质总量和种类二方面均较凹侧蛋白样品显著增加。(?)凸侧样品鉴定出103种蛋白,凹侧鉴定出55种蛋白,其中凸、凹侧共有的蛋白45种,凸侧特有蛋白58种,凹侧特有蛋白10种。(?)在凸侧发现的蛋白质中发现至少3种具有促进细胞和组织成熟、分化功能的蛋白质;而凹侧特有蛋白质中发现一种非常重要的生长抑制因子。结论:(?)第一次对人类脊柱软骨终板进行蛋白质组分析,得到AIS患者的凸、凹侧蛋白质组(?)AIS患者侧凸顶点软骨终板凸、凹侧蛋白质组存在明显差异。(?)蛋白质表达差异与其椎间盘的不对称发育密切相关。(?)引起椎间盘不对称发育的关键蛋白有待于进一步研究来寻找更多还原

【Abstract】 BackgroundAdolescent idiopathic scoliosis(AIS) is the most common form of spinal deformity of teenagers.The incidence of AIS among adolescents is approximately 3% as identified by a Cobb angle≥10°.AIS falls within the subcategory of "late-onset" idiopathic scoliosis which is defined as diagnosis at age≥10 years of age as opposed to "early onset" which can include infantile and juvenile cases.It is the most common form of idiopathic scoliosis accounting for between 80-85%of cases.Diagnostic criteria include:age≥10 years,Cobb angle of≥10°and exclusion of neuromuscular or congenital etiologies.Clinically AIS can be screened for using the Adams forward bend test and quantifying the degree of trunk rotation using a scoliometer,while the radiographic determination of the Cobb angle remains the gold standard.AIS may progress to result in pain,cardiopulmonary dysfunction,and dissatisfaction with appearance.More severe curves and progressive curves may require treatment including bracing or surgery.AIS has no validated etiology and occurs in otherwise healthy adolescents. Several hypotheses have been brought forward to explain the etiology of AIS, including genetic,biochemical,mechanical,neurological,muscular,hormonal,and even environmental factors.Twin studies have shown a much higher incidence of disease among monozygotic twins compared to dizygotic twins,lending credibility to a genetic component to the disease. Controversy,however,exists as to the exact mode of inheritance.Recent research has mapped genetic loci of AIS to chromosomes 9,17 and 19,but the inheritance pattern is not clear.Other research has failed to demonstrate a role for the growth hormone receptor gene as a predisposing gene or disease modifier gene in AIS.Asymmetrical interverteberal disc formation and growth is the structural foundation of deformity and disease progression in AIS patients.It has been shown that the convex side of interverteberal discs are found well developed compared to the concave side,both on radiographs and during anterior correction surgery procedures.The difference in histological grades and cellular activity between the convex and concave side indicated that the growth of AIS patients have different growth kinetics in endplate cartilage which may affect the curve progression. Ultimately,the differential growth in the endplates must result from some origin factors leading to differential expression of proteins.New advances in proteomics bio-technology now make it possible to directly analyze those proteins from harvested cartilage tissues.The pathogenesis of AIS is still unknown yet.Many different theories exist for the etiology.This study is the first major proteomic investigation of interverteberal disc cartilage.The proteins found with significant asymmetry in expression patterns may be implicated as causative of deformity,or may be the result of differences in the biomechanical environment within the disc,and secondary epiphenomena.Objective:To identify protein expression in asymmetrically developed intervertebral discs of adolescent idiopathic scoliosis(AIS) patients and try to find out factors that may originate or strengthen the situation.Methods:The apex intervertebral discs were harvested from 6 AIS patients during anterior correction surgery.Protein extraction were performed and we used shotgun strategy (2D-LC-MS/MS analysis) to achieve proteomics investigation.MS and tandem mass spectra were extracted from the XCalibur data system format(.RAW) into DTA format by Extact_msn.Tandem mass spectra were interpreted by SEQUEST against human ref protein database.CILP and HSP27 were performed Western Blot procedure to testify the mass spectra results.Results:The consistency of the extracted protein was tested through Bradford procedure[23], and the average concentration is 3.02mg/ml(2.20-3.97mg/ml).SDS-PAGE was performed to collect major complexion of the protein samples.Both quantity and concentration of the proteins from convex side were found to be higher than that from the opposite side.One hundred and three proteins were identified from samples of the convex side and fifty-five from the concave side,forty-five proteins were found both in convex side and concave side,while 58 and 10 proteins were found only in the convex side versus the concave side respectively.Conclusions:This is the first proteomic characterization of endplate cartilage phenotypes.Different protein expression levels between the convex and concave sides of the intervertebral discs endplate cartilage in AIS patients were indentified by shot-gun proteomics strategy.Though the result is preliminary,and it is still not clear that these differences are original factors of AIS or secondary cellular responses to mechanical compression and tension on the intervertebral disc at the apex of the curve. The study sheds new light on the genetic pathogenesis of adolescent idiopathic scoliosis and other disc related spinal diseases.The future directions identified appear promising.(For example in vitro mutation analysis studies could help in determining how these proteins affect cartilage tissue and AIS progress) Molecular and genetic mediations could help on finding how these proteins affect cartilage tissue and AIS progress.更多还原