摘要
[背景]
脊柱侧凸是常见的脊柱畸形,通常发生于青春发育期,女性多见,若不及时诊治,部分病人将会发展加重,引起躯体严重畸形,不但影响心肺功能、导致截瘫,还会给患儿心理健康造成严重危害。该病发病率由于诊断手段和标准的差异,一般认为在2%左右。在所有患者中,80%的患者病因不明,被称之为“特发性脊柱侧凸(Idiopathic Scoliosis,IS)",由于这种疾病常常发生于青少年,因此也叫青少年特发性脊柱侧凸(Adolescent Idiopathic Scoliosis,AIS)。尽管脊柱侧凸临床上已诊治多年但是发病机制尚不清楚。针对脊柱侧凸的病因,近年来,国内外就其遗传、生长发育、神经肌肉、激素、结缔组织等方向做了大量的研究。尤其是遗传基因的研究最为热门,国内外学者在IS的遗传模式和易感基因方面均取得了一些列的进展,但目前为止,还没有哪一个研究成果能够真正被学术界广泛接受。最近有报道,核因子-kappaB (NF-κB)信号通路在调节细胞的生长发育方面、细胞凋亡等方面同样起着重要作用。该因子是细胞中一个重要的转录调节因子,静息状态下与IκB相结合形成复合体,以无活性状态存在于胞浆中。当细胞受到外界因素刺激时,NF-κB暴露出核定位信号(NLS)进入核内,使其具有生物活性。目前研究发现NF-κB在褪黑素的合成、调节等方面均产生作用,并且相互影响,其初步研究符合特发性脊柱侧凸的临床特征。
[目的]
人们不能完全解释其确切发病原因,实际上“脊柱侧凸”只是一个症状诊断,短期内对该疾病进行病因诊断为时尚早,还需要大量的工作。结合临床实际观察特发性脊柱侧凸患者同时具有其他异常的比例也很高,如：神经系统异常、生长发育快速、三尖瓣返流、骨质缺乏等等。因此,本人推测特发性脊柱侧凸可能是一个全身性疾病的局部表现。同时NF-κB在细胞的生长、凋亡以及应激因子的释放等方面具有重要的调节作用,如果NF-κB信号通路异常,将导致全身性的异常,这种结果与特发性脊柱侧凸合并的其他表现类似。对褪黑素与核因子KB之间的关系及其调控因素的研究可能是解释IS发病机制的突破口。本课题拟在前期工作的基础上,在分子生物学水平,从信号传导角度,深入研究核因子KB在特发性脊柱侧凸发病中的作用、相互关系和机制,不但能进一步加深对特发性脊柱侧凸病因的认识,也可以为临床从根本上预防特发性脊柱侧凸的发生发展提供理论基础和新的治疗策略。若该类研究成果能最终应用于临床,将可以降低发病率,从源头上控制病情,减轻社会和家庭的负担,降低医疗支出,增加生产力和社会效益。
[方法]
1.临床研究
以特发性脊柱侧凸和非特发性脊柱侧凸患者(如先天性脊柱侧凸或神经纤维瘤病性脊柱侧凸)为对象进行研究。
(1)临床患者NF-κB活性的检测：抽血取样,并于前路和后路手术取脊柱骨(椎板、棘突、椎体终板)和软组织(椎间盘、各种韧带、椎旁肌)标本,比较不同类型脊柱侧凸患者脊柱两侧的NF-κB浓度及NF-κB p65亚基mRNA表达水平。
(2)观察临床患者褪黑素水平及其受体基因表达与NF-κB之间的关系：包括夜间褪黑素水平检测以及通过术中取材检测患者不同部位的褪黑素受体mRNA表达水平,并将结果与NF-κB检测结果进行综合分析。
(3)观察上述检测结果与患者临床指标进行相关性分析。患者的侧凸类型(特发性及非特发性)、形态(弯曲发生的不同部位：胸弯、腰弯、胸腰段弯曲、胸腰椎双主弯等)、柔韧度、严重程度、进展速度等临床指标进行相关性研究。
2.动物实验
以鸡特发性脊柱侧凸动物模型为研究对象进行研究。
(1)制作动物模型
切除雏鸡(孵化3天的小鸡)松果体建立特发性脊柱侧凸动物模型；
(2) NF-κB活性及含量的检测
检测范围包括不同时段内白细胞、脊柱骨组织(椎板、棘突、椎体终板)和软组织(脊髓、椎间盘、各种韧带、椎旁肌)标本的NF-κB浓度及NF-κB p65亚基mRNA表达水平。
(3)观察褪黑素水平及其受体基因表达与NF-κB之间的关系：
按照对照组、切除松果体已发生脊柱侧凸和未发生脊柱侧凸的动物进行分组,测量其夜间褪黑素水平检测以及不同部位的褪黑素受体mRNA表达水平,并将结果与NF-κB检测结果进行综合分析。探讨褪黑素与NF-κB在鸡脊柱侧凸发生中的相互联系。
[结果]
临床研究
1.临床患者NF-κB活性的检测
(1)轻中度(Cobh<60°)IS患者外周血、椎板、椎旁肌中NF-κB活性同非特发性侧凸及正常组相比无明显差别。
(2)重度(Cobb≥60°)IS患者外周血、椎板中NF-κB活性同非特发性侧凸及正常组相比无明显差别；但椎旁肌中NF-κB活性升高。
(3)RT-PCR进一步验证：重度(Cobb≥60°)IS患者椎旁肌中NF-κB活性较之非特发性侧凸及正常组升高。
(4)重度(Cobb≥60°)IS患者凹侧椎旁肌中NF-κB活性较凸侧高。
2.临床患者褪黑素水平及其受体基因表达与NF-κB之间的关系
(1)轻中度(Cobb<60°)IS患者外周血、椎板、椎旁肌中褪黑素水平同非特发性侧凸及正常组相比无明显差别。
(2)重度(Cobb≥60°)IS患者外周血、椎旁肌中褪黑素浓度较之非特发性侧凸及正常组低；但在椎板中无明显差异。
(3)Western-blot验证进一步说明：重度(Cobb≥60°)IS患者椎旁肌中褪黑素浓度较之非特发性侧凸及正常组低。
(4)重度(Cobb≥60°)IS患者椎旁肌中褪黑素水平与NF-kB活性呈负相关,但外周血中无此相关性。
3.上述检测结果与患者临床指标进行相关性分析
(1)重度(Cobb≥60°)IS患者椎旁肌中褪黑素水平与胸弯型、侧凸进展、侧凸严重度具有相关性。
(2)重度(Cobb≥60°)IS患者椎旁肌中NF-κB水平与胸弯型、侧凸进展、侧凸严重度同样具有相关性。研究结果提示：褪黑素及NF-κB在侧凸发生过程中的作用尚不明确,但在其发展过程中起重要作用。
动物实验
1.建立脊柱侧凸模型
切除雏鸡(孵化3天的小鸡)松果体建立特发性脊柱侧凸动物模型；本研究动物模型成功建立,松果体切除组有65.4%出现侧凸(Cobb>10°)。术后发生侧凸畸形的鸡,畸形种类多种多样,且术后3月,脊柱侧凸角度进一步加重。
2. NF-κB活性及含量的检测
发生侧凸的鸡2周时血中的NF-κB浓度较对照组、未发生侧凸的鸡高；3个月时监测：血、椎板中浓度同对照组无明显差异,椎旁肌中的NF-κB浓度较对照组、未发生侧凸的鸡高。
3.观察褪黑素水平及其受体基因表达与NF-κB之间的关系：
2周时夜间褪黑素水平在切除松果体已发生脊柱侧凸和未发生脊柱侧凸的两组动物中均明显降低,较对照组差异明显；3个月时,各种组织中褪黑素水平仍较低。
研究结果提示：血中、骨组织中可能有其他因素参与代偿机制,使得NF-κB活性恢复正常,也提示上述两种因子可能对椎旁肌的影响最大。
[结论]
本研究针对脊柱侧凸这一世界难题,以临床特发性脊柱侧凸患者和雏鸡脊柱侧凸动物模型为研究对象,采用分子生物学研究方法,以NF-κB信号传导途径为研究靶点,(1)观察了临床特发性脊柱侧凸患者血液及骨及软组织NF-κB活性、褪黑素水平及其受体基因表达、并将组织学结果与患者临床指标进行相关性分析；(2)切除雏鸡松果体建立特发性脊柱侧凸动物模型,检测其组织NF-κB活性及含量、褪黑素水平及其受体基因表达、并将组织学结果与患者临床指标进行相关性分析。
结果显示：(1)特发性脊柱侧凸发生机制复杂,多因素作用的结果；(2)褪黑素及NF-κB在侧凸发生过程中的作用不明确；(3)褪黑素及NF-κB在侧凸的发展过程中起着重要的作用；(4)褪黑素及NF-κB的重要作用位点是椎旁肌；(5)需要进一步行基因水平的研究以阐述其机制。
本课题拟在前期工作的基础上,在分子生物学水平,从信号传导角度,深入研究核因子KB在特发性脊柱侧凸发病中的作用、相互关系和机制,不但进一步加深了对特发性脊柱侧凸病因的认识,也可以为临床从根本上预防特发性脊柱侧凸的发生发展提供理论基础和新的治疗策略。
[Backgrounds]
Scoliosis is a disorder that causes an abnormal curve of the spine, or backbone.The spine has normal curves when looking from the side, but it should appearstraight when looking from the front. Scoliosis is about two times morecommon in girls than boys. It can be seen at any age, but it is most commonin those over10years of age. Scoliosis is hereditary in that people withscoliosis are more likely to have children with scoliosis; however, there is nocorrelation between the severity of the curve from one generation to the next.Scoliosis can affect about2%of females and0.5%of males. In most cases,the cause of scoliosis is unknown (idiopathic). This type of scoliosis isdescribed based on the age when scoliosis develops. If the person is less than3years old, it is called infantile idiopathic scoliosis. Scoliosis that developsbetween3and10years of age is called juvenile idiopathic scoliosis, andpeople that are over10years old have adolescent idiopathic scoliosis. Morethan80%of people with scoliosis have idiopathic scoliosis, and the majorityof those are adolescent girls. Scoliosis was medical diagnosis and treatmentaboutr many years, but the pathogenesis is not clear now. The etiology ofscoliosis in recent years, about genetic, neuromuscular, hormonal,connective tissue, there have a lot of research about those. Especially geneticresearch is the most popular in the IS, but so far, there have no report andresearch were accepted by academics. Recently, nuclear factor-kappa B(NF-κB) signaling pathway in cell growth and development, plays animportant role in cell apoptosis. This factor is an important transcriptionalregulator in the cell factor, resting with IκB combine to form the compositebody, exists in an inactive state in the cytoplasm. When cells stimulated byexternal factors, NF-κB expose the nuclear localization signal (NLS) into thenucleus, so that it has a biological activity. The current study found thatNF-κB in melatonin synthesis, regulate effect and influence each other, andpreliminary studies met the clinical features of idiopathic scoliosis
[Objectives]
People can not explain the pathogenesis of scoliosis, in fact, the “scoliosis” isa symptom diagnosis, we need work hard to know the pathogenesis of scoliosis. The scoliosis patients always have another disease. Such as: thenervous system abnormalities, rapid growth and development, tricuspidregurgitation, osteopenia. Therefore, I speculated that idiopathic scoliosis maybe a local manifestations in systemic diseases. NF-kappaB has an importantregulatory role in cell growth, apoptosis, and the release of the stress factor,NF-kappaB signal pathway is abnormal, will lead to systemic abnormalities,such results idiopathic scoliosis the combined performance is similar. Theresearch on the relationship between melatonin and nuclear factor kappa Band its regulatory factors may explain the pathogenesis of IS.
[Methods]
1. Clinical research
Patients with scoliosis, idiopathic scoliosis and unspecified (such ascongenital scoliosis or neurofibromatosis scoliosis) as the object.
1.1Clinical patients with NF-κB activity detection:
Blood sampling, and take in the anterior and posterior surgery spine bone(lamina, spinous process, vertebral endplate) and soft tissue (intervertebraldisc, the various ligament, paraspinal muscles) specimens, comparingdifferent types of scoliosis patients with spinal both sides of the concentrationof NF-κB and NF-κB p65subunit mRNA expression levels.
1.2Observed clinical patients melatonin levels and its receptor relationshipbetween gene expression and NF-κB:
Including nighttime melatonin levels detected based detection of patients indifferent parts of the melatonin receptor mRNA expression through thesurgery, the results of a comprehensive analysis of the test results of theNF-κB.
1.3Observed correlation analysis of the test results and clinical indicators.
Patients with scoliosis type (idiopathic, unspecified onset), morphology(bending different parts: thoracic curve, lumbar curve, thoracolumbar bending,thoracolumbar double major curve, etc.), flexibility, severity, rate of progresscorrelation studies and other clinical indicators.
2Animal experiments
Chicken idiopathic scoliosis animal model for the study of objects.
2.1Make animal model
Resection chicks (3days to hatch chicks) pineal establish idiopathic scoliosisanimal model;
2.2NF-κB activity and content detection
Detection range including different times in a day, white blood cells, the spinebone tissue (lamina, spinous process, vertebral endplate) and soft tissue(spinal cord, intervertebral discs, various ligaments, paraspinal muscles)specimens of the concentration of NF-κB and NF-κB p65sub-based mRNAexpression levels.
2.3Observed melatonin levels and its receptor relationship between geneexpression and NF-κB:
Removal of pineal scoliosis occurs in accordance with the control group, andthe animals did not occur scoliosis packet measurement detection, anddifferent parts of the night-time melatonin levels of melatonin receptormRNA, and the results of NF The test results-κB comprehensive analysis.Explore melatonin NF-κB chicken scoliosis occurs linkages.
2.4Antisense oligonucleotide of NF-κB in the regulation of NF-κB activity:
Idiopathic scoliosis animal model for the study, the intervention of NF-κBactivity of NF-κB antisense oligonucleotides to observe the effects of itsregulation of NF-κB activity.
[Results]
Clinical Research
1. Clinical patients with NF-κB activity detection
1.1Middle level (Cobb <60°) IS peripheral blood of patients, laminectomy,paraspinal muscle activity of NF-κB was no significant difference comparedwith nonatopic onset scoliosis and normal group.
1.2Severe (cobb≥60 ) IS in peripheral blood of patients with NF-κBactivity in the lamina was no significant difference compared with nonatopiconset scoliosis and normal group; paraspinal muscles in NF-κB activity iselevated.
1.3RT-PCR validation: severe (cobb≥60 ) IS paraspinal muscles inpatients with NF-κB activity increased compared to the the Sofitel onsetscoliosis and normal group.
1.4Severe (cobb≥60 ) IS patients concave side than the convex side of paravertebral muscle in NF-κB activity.
2The relationship between clinical melatonin levels in patients and itsreceptor gene expression and NF-κB
2.1(cobb <60°) IS, mild to moderate peripheral blood of patients, thelamina, paraspinal muscles melatonin levels was no significant differencecompared with nonatopic onset scoliosis and normal group.
2.2Severe (cobb≥60 ) IS peripheral blood of patients the paraspinalmuscles melatonin concentration is low compared with the the Sofitel onsetscoliosis and normal group; However, no significant difference in the lamina.
2.3Western-blot validation further explanation: severe (the cobb≥60 ),the IS patients paraspinal muscles in melatonin concentration is low comparedwith the the Sofitel onset scoliosis and normal group.
2.4Severe (cobb≥60 ) the IS patients paravertebral muscle inmelatonin level was negatively correlated with the NF-kB activity, but nosuch correlation in the peripheral blood.
3. Correlation analysis of the test results and clinical indicators
3.1Severe (cobb≥60 ) the IS patients paraspinal muscles in melatoninlevels and thoracic curve type, curve progression, scoliosis severitycorrelation.
3.2Severe (cobb≥60 ) the IS paraspinal muscles in patients withNF-κB level, thoracic curve type, curve progression, scoliosis severity samecorrelation. The results suggest that: melatonin and NF-κB role in the processof scoliosis occurs is not clear, but play an important role in the developmentprocess.
Animal experiments
(1) Establishment of scoliosis modelResection chicks (3days to hatch chicks) pineal this study successfullyestablished animal models to establish an animal model of idiopathic scoliosis;pinealectomy group,65.4%had scoliosis (Cobb>10°). Postoperative thescoliosis chicken variety of deformities, and3months after surgery, scoliosisangle further aggravated.
2. NF-κB activity and content detectionConcentration of NF-κB in the blood when the occurrence of scoliosis chickens two weeks compared with the control group, the not occur scoliosischicken;3months monitoring: blood, lamina concentration with the controlgroup, no significant difference in the paraspinal muscle NF-κB concentrationcompared with the control group, the not occur scoliosis chicken.
3. Observed melatonin levels and its receptor relationship between geneexpression and NF-κB:Two weeks when nighttime melatonin levels haveoccurred in the removal of pineal were significantly lower in the two groupsof animals scoliosis and scoliosis, significant differences compared to thecontrol group;3months, a variety of organizations melatonin The level wasstill low.The study results suggest that: the blood, the bone tissue, there may be otherfactors involved in compensatory mechanisms, NF-κB activity returned tonormal, and also prompted these two factors may affect the paraspinalmuscles.
[Conclusions]
Scoliosis is a serious problem in the world, and clinical idiopathic scoliosisand the chicks spinal scoliosis animal model for the study of molecularbiology research methods in the study of NF-κB signaling pathway.(1)Observation of clinical idiopathic scoliosis patients with blood and bone andsoft tissue NF-κB activity, melatonin levels and its receptor gene expressionand histological results of correlation analysis and clinical indicators;(2)Idiopathic scoliosis animal model, NF-κB activation, tissue sections, andcontent) removal of the chick pineal melatonin levels and its receptor geneexpression and histological results with clinical indicators correlation analysis;(3) Idiopathic scoliosis animal model for the study of detection of NF-κBantisense oligonucleotide intervention of NF-κB activity.The results:(1) idiopathic scoliosis`s mechanism is very complex, the resultof multiple factors;(2) The melatonin and NF-κB role in the process ofscoliosis occurs is not clear;(3) Melatonin and NF-κB plays an important rolein the development process of the scoliosis;(4) Melatonin and the importantrole of NF-κB sites paraspinal muscles;(5) Need to further line gene levelstudies to illustrate its mechanism.
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73. Byrd JA. Current theories on the etiology if idiopathic scoliosis. Clin Orthop1998;229:114-9.
74. Cowell HR, Hall JN, MacEwen GD. Genetic aspects of idiopathic scoliosis. Clin Orthop1972;86:121-31.
75. Kazmin AI, Merkorieva RV.. Role of disturbance of glucosaminoglycane metabolism in thepathogenesis of scoliosis. Orthop Traumatol Protez1971;32:87-91.
76. Kindsfater K, Lowe T, Lawellin D, et al. Levels of platelet carmodulin for the prediction ofprogression and severity of adolescent idiopathic scoliosis. J Bone Joint Surg [Am]1994;76:1186-92.
77. Koulu M, Lammintausta R. Effect of melatonin on L-tryptophan and apomorphine stimulatedgrowth hormone secretion in man. J Clin Endocrinol Metab1979;49:70-2.
78. Lewy AJ, Tesuo M, Markey SP, et al. Pinealectomy abolishes plasma melatonin in the rats. JClin Endocrinol Metab1980;50:204-5.
79. McInnes E, Hill DL, Raso VJ, et al. Vibratory response in adolesencts who have idiopathicscoliosis. J Bone Joint Surg [AM]1965;73:1208-12.
80. Mori S. Contribution of postural muscle tone to full expreeion of posture and locomotormovements: Multi-faceted analyses of its setting brainstem-spinal cord mechanisms in cat. JpnJ Physiol1989;39:785-809.
81. Nachemson A, Sahlstrad T. Etiologic factors in adolescent idiopathic scoliosis. Spine1977;2:176-84.
82. Ozaki Y, Lynch HJ. Presence of melatonin in plasama and urine of pinealectomized rats.Endocrinol1976;99:641-4.
83. Sahstrad T, Ortengren R, Nachemson A. Postural equilibrium in adolescent idiopathic scoliosis.Acta Orthop Scand1978;49:354-65.
84. Schultz AB. A biomechanical view of scoliosis. Spine1976;1:162-71.
85. Skogland LB, Miller JAA,. Growth-related hormones in idiopathic scoliosis. Acta OrthopScand1980;51:779-89.
86. Smythe GA, Lazarus L. Growth hormone regulation by melatonin and serotonin. Nature1973;224:230-1.
87. Thilland Mj. Deformation de la collone vertebrale consecutives al’epiphysictomie chez lepoussin. Extrait des comptes rendus de I’Association des Anatomistes1959;46:22-6.
88. Trouillas P, Brudon F, Adeleine P. Improvement of cerebellar ataxia with levoratory form of5-hydroxyterptophan: A double-bland study with quantified data processing. Arch Neurol1988;45:1217-22.
89. Wyatt MP, Barrack RL, Mubarak SJ, et al. Vibratory response in idiopathic scoliosis. J BoneJoint Surg [Br]1968;50:24-30.
90. Yamamoto H, Tani T, MacEen GD, et al. An evaluation of brainstem function as aprognostication of early idiopathic scoliosis. J Pediatr Orthiop1982;2:521-7.
91. Yarom R, Robin GC. Studies on spinal and peripheral muscle from patients with scoliosis.Spine1979;4:12-21.