【作者】 张臻；

【导师】 邱勇；

【作者基本信息】 南京大学 ， 外科学， 2017， 博士

【摘要】 第一章青少年特发性脊柱侧凸可溶性瘦素受体水平异常的机制研究背景青少年特发性脊柱侧凸(adolescent idiopathic scoliosis,AIS)是最常见的三维脊柱畸形,最常累及10-16岁的青春期女孩,若未及时发现和干预,脊柱侧凸可不断加重以致影响呼吸、循环功能,甚至可导致患者死亡。然而目前关于AIS的病因和发病机制仍不明确。既往研究发现AIS患者具有异常的生长特征。AIS患者与正常同龄青少年相比具有较高的校正身高和生长高峰速度、较低的体重和体质量指数(bodymassindex,BMI)以及较较低的骨密度,而这种异常生长特征被认为可能与AIS的发生发展相关。诸多学者都提出假说试图解释AIS患者的这种异常生长特征,其中瘦素代谢异常假说获得了广泛关注。瘦素是一种调节青春期女孩生长发育的一种重要的激素,主要由脂肪组织分泌,可穿透血脑屏障与下丘脑的瘦素受体结合,通过负反馈调节机制调控骨和脂肪组织的生长代谢。AIS患者的这些异常生长特征高度提示可能存在瘦素代谢调节系统的异常。可溶性瘦素受体(soluble leptin receptor,sOB-R)是调控瘦素生物利用度和跨血脑屏障运输的关键因素之一,它是由跨细胞膜瘦素受体(主要为OB-Ra和OB-Rb)的膜外部分经去整合素金属蛋白酶(A Disintegrin and Metalloproteinase,ADAM)水解后溶于外周血后形成。sOB-R可通过结合游离瘦素调节外周痩素的生物利用度,并可促进瘦素跨血脑屏障运输,从而增强中枢瘦素的活性。既往研究显示,AIS患者外周血sOB-R水平高于正常对照,且游离瘦素指数(血清瘦素/可溶性瘦素受体水平,FLI,反应瘦素生物利用度)则低于正常对照,高度提示AIS患者存在因sOB-R水平异常导致的瘦素生物利用度异常,并且这种瘦素生物利用度异常可能是AIS患者异常生长特征和脊柱侧凸发生发展的重要原因。然而,目前对于AIS患者外周血sOB-R水平异常的机制仍缺乏研究。由于ADAM是调节sOB-R生成的关键酶,因此本研究将进一步探究ADAM是否在AIS外周血sOB-R水平异常中发挥作用。第一节可溶性瘦素受体生成机制的研究目的:通过构建OB-Ra和OB-Rb过表达稳转细胞株探究与生成sOB-R相关的ADAM的亚型。方法:首先构建人瘦素受体(OB-Ra和OB-Rb)过表达慢病毒载体,然后将慢病毒载体转入表达各种ADAM亚型的293T细胞中,从而建立OB-Ra和OB-Rb过表达稳转细胞株。然后将过表达稳转细胞株分组,分别加入ADAM10抑制剂GI254023X(GI)、ADAM10 和 ADAM17 抑制剂 GW280264X(GW),通过检测并比较各组间细胞培养液上清中sOB-R的浓度,确认与sOB-R生成相关的ADAM亚型。结果:1.OB-Ra和OB-Rb过表达细胞株荧光表达率在80-90%以上,均能高水平合成相应瘦素受体亚型mRNA,稳转细胞株构建成功;2.OB-Ra过表达细胞GI组和GW组培养24小时后培养基中sOB-R含量较对照组分别下降45.0%和51.4%,培养48小时后较对照组分别下降52.1%和61.0%。3.OB-Rb过表达细胞GI组和GW组培养24小时后培养基中sOB-R含量较对照组分别下降38.5%和36.8%,培养48小时后较对照组分别下降46.2%和51.1%。结论:OB-Ra和OB-Rb过表达稳转细胞均能高表达相应瘦素受体亚型,稳转细胞株构建成功。跨膜瘦素受体水解生成可溶性瘦素受体依赖于ADAM蛋白酶,其中ADAM 10发挥主要作用,ADAM 17发挥次要作用。第二节青少年特发性脊柱侧凸去整合素金属蛋白酶水平与异常可溶性瘦素受体水平的关系目的:评估AIS患者体内ADAM10和ADAM17的表达情况,从上游探究AIS sOB-R水平升高的原因,从而进一步阐明瘦素生物利用度异常与脊柱侧凸病因学之间的关系。方法:收集30例AIS患者和30例同龄正常青少年的外周血,提取白细胞中RNA,通过 Real-time PCR 方法检测 OB-Ra、OB-Rb、ADAM10 和 ADAM17 mRNA的合成水平,并通过酶联免疫吸附测定法(Enzyme Linked ImmunoSorbent Assay,ELISA)检测AIS患者和对照组血清中瘦素、sOB-R、ADAM10和ADAM17的含量,比较AIS患者和同龄正常青少年之间是否存在差异。结果:AIS患者血清瘦素水平显著低于正常对照组(7.3 ± 4.8 ng/ml vs.8.7 ± 4.2 ng/ml,P=0.044),sOB-R 水平显著高于正常对照组(26.8 ±6.9 ng/mlvs.22.5 士6.1 ng/ml,P = 0.012)。AIS患者ADAM10 mRNA合成水平和血清含量均显著高于对照组(P<0.05),而ADAM17的mRNA合成水平和血清含量与对照组无显著差异。结论:AIS患者的外周血sOB-R含量和ADAM10表达水平均显著高于正常青少年,而跨膜瘦素受体表达水平与正常青少年无显著差异,AIS患者高sOB-R水平可能是由ADAM10表达升高引起。第二章脊柱畸形相关临床研究第一节Lenke 1型青少年特发性脊柱侧凸后路矫形术后颈椎矢状面最终形态的预测因素:一项长期随访研究目的:1.评估Lenke 1型AIS患者行后路矫形手术术前、术后即刻和末次随访时的颈椎矢状面形态,分析Lenke1型患者术前、术后的颈椎矢状面代偿模式。2.寻找可以预测AIS最终(末次随访)颈椎矢状面形态的术前或术后即刻的影像学参数。方法:回顾性分析44例于本院脊柱外科行后路矫形手术、术后随访至少5年的Lenke1型AIS患者。收集所有患者的术前、术后即刻和末次随访的影像学和临床资料。在站立位全脊柱正侧位X线片上测量以下影像学参数:胸主弯Cobb角(Main thoracic curve,MTC)、上胸弯 Cobb 角(Proximal thoracic curve,PTC)、颈椎前凸角(Cervical lordosis,CL)、颈椎矢状垂直偏距(Cervical sagittal vertical axis,CSVA)、T1 倾斜角(T1 slope,T1S)、上胸椎后凸角(Upper thoracic kyphosis,UTK)、主胸椎后凸角(Main thoracic kyphosis,MTK)、全胸椎后凸角(Globalthoracickyphosis,GTK)、腰椎前凸角(Lumbarlordosis,LL)、骶骨倾斜角(Sacrum slope,SS)、骨盆入射角(Pelvicincidence,PI)、骨盆倾斜角(Pelvictilt,PT)。通过Pearson相关性分析、逐步多元线性回归分析和受试者工作特征曲线(Receiver operator characteristic,ROC)寻找可以预测最终(末次随访)颈椎矢状面形态的术前和术后即刻的影像学参数。结果:44例Lenke 1型AIS患者CL由术前的-6.6°± 8.8°改善为术后即刻的-3.8°±8.7°,并在末次随访时改善为0.5°±7.3°。CSVA术后减小,但差异未达统计学意义。35例(79.5%)患者术前颈椎矢状面形态表现为后凸或曲度变直,其中9例(25.7%)患者在末次随访时获得了前凸的颈椎矢状面形态;9例(20.5%)患者术前颈椎矢状面形态表现为前凸,且在末次随访时均维持前凸状态。Pearson相关性分析表明术前CL仅与T1S和UTK相关,而术后CL与T1S、UTK和GTK均相关。多元线性回归分析得到最终CL的预测方程:最终CL =-2.80+ 0.51 ×术前CL + 0.53 ×术后即刻TIS。ROC曲线显示术前CL ≥-4.5°和术后即刻T1S ≥ 11.3°是最终颈椎矢状面形态为前凸型的预测因素。结论:随着后路脊柱畸形矫形术后整体和胸椎局部的矢状面形态的重建,Lenke 1型AIS患者的CL显著增大,颈椎矢状面形态显著改善。由于手术的干预,术前和术后的颈椎矢状面代偿模式存在差异。对于Lenke1型AIS患者,术前CL和术后即刻T1S可作为预测最终颈椎矢状面形态的预测因素。第二节硬膜外脂肪增多症一一一个易忽视的脊柱后凸畸形继发性椎管内异常目的:验证如下猜想:SEL是各种脊柱后凸畸形常见的继发性椎管内异常。方法:回顾性分析16例先天性脊柱后凸(Congenitalkyphosis,CK)患者、40例休门氏病(Scheuermann kyphosis,SK)患者、13例结核性脊柱后凸(Tuberculotic kyphosis,TK)患者,以及69例年龄和性别匹配的正常对照的临床和影像资料。记录患者的体质量指数(body mass index,BMI),在全脊柱侧位X线片上测量后凸Cobb角。在全脊柱矢状面MRI上测量以下参数:顶椎区硬膜外脂肪矢状径(EFA)、硬膜囊矢状径(DSA),以及非后凸区硬膜外脂肪矢状径(EFN)、硬膜囊矢状径(DSN)。分别计算顶椎区和非后凸区的硬膜外脂肪比(EFR):EF/(EF + DS)。结果:CK组、SK组和TK组患者的EFA和和EFRA显著高于正常对照组(P<0.05)。7 例 CK 患者(43.8%),8 例 SK 患者(20.0%),和 11 例 TK 患者(84.6%)达到SEL的诊断标准,而正常对照组仅分别有1例(6.3%)、1例(2.5%)和 0 例(0%)达到 SEL 诊断标准(P = 0.019,0.014,<0.001)。Spearman相关性分析显示,在三组患者中,脊柱后凸Cobb角与硬膜外脂肪量均存在显著相关性。结论:SEL是各种脊柱后凸畸形常见的继发性椎管内异常。由于过多的硬膜外脂肪可压迫脊髓产生神经损害,因此脊柱外科医生应重视该现象。第三节Chiari畸形Ⅰ型后颅窝减压术后颈椎管再塑形目的:评估Chiari畸形Ⅰ型(Chiari malformation type Ⅰ,CMI)伴脊髓空洞患者后颅窝减压术(posterior fossa decompression,PFD)后颈椎管直径和锥度是否改变,并分析其与空洞缓解是否相关。方法:回顾性分析39例于本院脊柱外科行PFD手术、随访至少一年的CMI伴脊髓空患者的影像学资料。将患者分为PFD术后脊髓空洞改善组(syrinx-improved,SI 组)和脊髓空洞未改善组(syrinx-unimproved,SU 组),SI 组共有30例患者,SU组共有9例患者。在T2加权相MRI正中矢状位扫描层面上测量C2-7节段颈椎管前后径,以椎体节段为横坐标椎管前后径为纵坐标绘制散点图,利用Excel软件绘制二元回归方程趋势线,该趋势线的斜率记为颈椎管锥度(mm/节段)。比较术前和术后以及SI组和SU组间颈椎管前后径和椎管锥度是否存在差异。结果:在SI组中,PFD术后各节段颈椎管前后径均减小,其中C2、C3、C4、C6和C7减小程度达到统计学意义(P = 0.001,0.011,0.028,0.047和0.045),而在SU组中,各节段术前、术后颈椎管前后径均无显著变化。SI组C2-4节段的椎管锥度从术前的-1.38 ± 0.78 mm/节段显著减小为术后的-1.13 ± 0.95 mm/节段(P=0.024),然而C4-7节段的椎管锥度则无显著改变(P=0.553)。而在SU组中,C2-4和C4-7节段的椎管锥度术前、术后均无显著改变(P= 0.867,0.980)。结论:PFD术后脊髓空洞缩小的CMI患者颈椎管前后径减小,提示颈椎管随着脊髓空洞的改善而再塑形。另外,该再塑形过程使颈椎管锥度减小,颈椎管锥度减小可改善脑脊液动力学并进一步促进脊髓空洞的缩小。更多还原

【Abstract】 Chapter 1 The mechanism of abnormal soluble leptin receptor level in adolescent idiopathic scoliosisBackground:Adolescent idiopathic scoliosis(AIS)is the most common three-dimensional spinal deformity which mainly affects the adolescent girls aged 10-16.If left untreated,the spinal deformity could develop continuously and the patient may die from respiratory and circulation failure.Despite decades of research,the etiology and pathogenesis of AIS remains largely unknown.Previous anthropometric studies have reported an abnormal growth pattern in AIS which was considered to play an important role in the pathogenesis of AIS.AIS patients are reported to have taller stature and peak height velocity,lower weight and body mass index(BMI)and systemic low bone mass.Many researchers put forward different theories to explain this abnormal growth pattern.Among them,the leptin theory has received wide concern.Leptin,a hormone excreted by adipose tissue,plays an important role in regulating energy expenditure,bone metabolism,body weight,and the hypothalamic-pituitary function.indicated that the abnormal leptin function may contribute to the development and progression of AIS.Soluble leptin receptor(sOB-R),generated by shedding of membrane-anchored leptin receptors,is the key to regulate the transmembrane transportation and the bioavailability of leptin.Previous studies showed that AIS girls have significantly higher serum sOB-R level and lower free leptin index(FLI,leptin/sOB-R,indicating the bioavailability of leptin)than age-matched healthy girls.These results highly indicated that the abnormal leptin bioavailability caused by abnormal sOB-R level may play an important role in the abnormal growth pattern and the pathogenesis of AIS.However,the mechanism of elevated sOB-R level in AIS remains unknown.As A Disintegrin and Metalloproteinase(ADAM)is the key enzyme regulating the generation of sOB-R,this study would further investigate the role of ADAM in the abnormal serum sOB-R level in AIS.Section 1 The mechanism of generation of soluble leptin receptorObjectives:To identify the ADAM family members that participate in the generation of sOB-RMaterials and Methods:We constructed stable cell lines overexpressing human leptin receptors(OB-Ra and OB-Rb)by constructing lentivirus vectors and transfecting them into 293T cells which express different ADAM family enzymes.Divide the cells into subgroups and add AD AM10 inhibitor GI254023X(GI),AD AM10 and AD AM17 inhibitor GW280264X(GW)into different subgroups.Identify the ADAM family members that participate in the generation of sOB-R by evaluating and comparing the sOB-R level of different subgroups.Results:1.The leptin receptor mRNA expression level was significantly higher in cell lines overexpressing human leptin receptors(OB-Ra and OB-Rb)than in controls.Stable cell lines overexpressing OB-Ra and OB-Rb were successfully constructed.2.In GI group and GW group of cell lines overexpressing OB-Ra,the leptin level decreased by 45.0%and 51.4%after incubation for 24h,and decreased by 52.1%and 61.0%after incubation for 48h.3.In GI group and GW group of cell lines overexpressing OB-Rb,the leptin level decreased by 38.5%and 36.8%after incubation for 24h,and decreased by 46.2%and 51.1%after incubation for 48h.Conclusion:The stable cell lines overexpressing OB-Ra and OB-Rb were successfully constructed.The generation of sOB-R mainly relies on ADAM 10 by shedding of membrane-anchored leptin receptors.Section 2 The relationship between ADAM level and abnormal soluble leptin receptor level in AISObjectives:To evaluate the ADAM 10 and ADAM17 levels in AIS patients and to clarify the mechanism of abnormal soluble leptin receptor level in AIS.Materials and Methods:OB-Ra,OB-Rb,AD AM10 and AD AM17 mRNA levels in white cells together with serum leptin,sOB-R,ADAM 10,and ADAM 17 concentrations were measured in 30 AIS girls and 30 age-matched healthy girls.AD AM10 and AD AM17 mRNA levels were measured by real-time PCR and serum sOB-R,AD AM10,and ADAM17 concentrations were measured by ELISA.Results:AIS girls had significantly lower leptin level and higher sOB-R level than healthy controls(7.3 ± 4.8 ng/ml vs,8.7 ± 4.2 ng/ml,P = 0.044;26.8 ± 6.9 ng/ml vs.22.5 ± 6.1 ng/ml,P = 0.012;respectively).The mRNA level and serum concentration of AD AM10 in AIS patients were significantly higher than in controls(P<0.05).The mRNA level and serum concentration of ADAM 17 was not significantly different between AIS patients and controls.Conclusion:The ADAM10 level and serum sOB-R concentration in AIS patients were significantly higher than in controls,indicating that elevated ADAM10 expression may be responsible for the high sOB-R level in AIS.Chapter 2 Clinical studies about spinal deformitiesSection 1 Predictors of ultimate postoperative cervical sagittal alignment in main thoracic adolescent idiopathic scoliosis:a long-term follow-up studObjectives:To access sagittal compensatory mechanism of the cervical spine in thoracic adolescent idiopathic scoliosis(T-AIS)before and after posterior spinal fusion and to identify preoperative or immediate postoperative radiographic parameters that can predict the ultimate cervical sagittal alignment(CSA)after long-term follow-up.Materials and Methods:A retrospective study was performed on 44 T-AIS patients treated with posterior spinal fusion and with at least 5 years of follow-up.Preoperative,immediate postoperative and latest follow-up radiographs were reviewed measuring main thoracic curve(MTC),main thoracic curve(MTC),cervical lordosis(CL),cervical sagittal vertical axis(CSVA),upper thoracic kyphosis(UTK),main thoracic kyphosis(MTK),global thoracic kyphosis(GTK),lumbar lordosis(LL),pelvic incidence(PI),pelvic tilt(PT),and sacral slope(SS).Pearson correlation analysis,stepwise multilinear regression analysis,and ROC curve were performed to define the relationship between ultimate CL and preoperative or immediate postoperative radiographic parameters.Results:CL significantly improved from 6.6°t8.8° kyphosis preoperatively to 3.8°±8.7°kyphosis immediate postoperatively and to 0.5°±7.3° lordosis at the latest follow-up.Pre-and postoperative CSVA showed no significant difference.Pearson correlation coefficient test showed that CL was only correlated to T1 slope and UTK before surgery,while it was correlated to T1 slope,UTK,and GTK after surgery.The following equation was developed to estimate the ultimate CL:ultimate CL =-2.80 + 0.51× Preop CL + 0.53 × Postop T1 slope.Furthermore,ROC curve showed that preoperative CL ≥-4.5° was strongly predictive and postoperative T1 slope ≥ 11.3° was moderately predictive of lordotic cervical spine after long-term follow-up.Conclusion:For Lenke type 1 AIS patients,cervical lordosis significantly increased after surgery with the restoration of the global and regional sagittal profile.The sagittal compensatory mechanism of the cervical spine before surgery is different from that after surgery.In these patients,preoperative CL and immediate postoperative T1 slope,could be predictors of the ultimate CSA after long-term follow-up.Section 2 Spinal epidural lipomatosis-an easily ignored secondary intraspinal disorder in spinal kyphotic deformitiesBackground:A previous study reported a high prevalence of spinal epidural lipomatosis(SEL)in patients with Scheuermann kyphosis(SK)and suggested that it may play a role in the pathogenesis of this disease.According to our observation,however,SEL occurs in other spinal kyphotic deformities as well.The aim of this study was to test the hypothesis that SEL commonly occurs in patients with different types of kyphotic deformities as a secondary intraspinal disorder.Materials and Methods:MR images of 16 patients with congenital kyphosis(CK),40 patients with SK,13 patients with tuberculotic kyphosis(TK),and 69 age-and sex-matched controls were retrospectively evaluated.The body mass index(BMI),kyphosis Cobb angle,and sagittal diameters of spinal epidural fat(EF)and the dural sac(DS)in the apical region(EFA,DSA)and non-kyphotic region(EFN,DSN)were measured.The EF ratios at the apical vertebral level(EFRA)and in the non-kyphotic region(EFRN)were calculated as EF/(EF + DS).Results:EFA and EFRA were significantly higher among patients with CK,SK,and TK than among controls(P<0.05).Seven CK patients(43.8%),8 SK patients(20.0%),and 11 TK patients(84.6%)fulfilled the diagnostic criteria for SEL,while only 6.3%,2.5%,and 0%of patients in the control groups did(P = 0.019,0.014,and<0.001,respectively).Spearman’s correlation analysis showed statistically significant correlations between the kyphosis Cobb angle and the amount of EF in all three patient groups.Conclusions:SEL is a common secondary intraspinal disorder in different types of kyphotic deformities,and surgeons should pay increased attention to this intraspinal anomaly because excessive EF may compress the spinal cord and cause neurological deficits.Section 3 Remodeling of the cervical spinal canal in patients with Chiari malformation type I after posterior fossa decompressionObjective:Previous research reported milder taper ratios of cervical spinal canals in patients with Chiari malformation type I(CMI)with distended syringes compared to those with nondistended syringes,indicating a reciprocal interaction between the cervical spine anatomy and the syrinx.This study aimed to determine whether the cervical spinal canal remodels in patients with CMI after posterior fossa decompression(PFD).Materials and methods:Thirty-nine patients with CMI-associated syringomyelia were divided into two groups:30 with an improved syrinx after PFD and 9 with unimproved syrinx after PFD.On a midline T2-weighted MR image,the anteroposterior diameters of the spinal canal were measured at C2-7 cervical levels,and a trend line was fitted by least squares regression.The slope of this line was recorded as the taper ratio.The diameters and the taper ratios were compared between the pre-and post-operative settings and between the two groups.Results:In the syrinx-improved group(group SI),the average diameter of the spinal canal at C2,C3,C4,and C7 levels significantly decreased after surgery(P = 0.001,0.011,0.028,and 0.045 respectively).Meanwhile,the taper ratios for C2-4 became milder from-1.38 ± 0.78 to-1.13 ± 0.95(P = 0.024).In contrast,neither the diameters nor the taper ratios changed significantly after PFD in the syrinx-unimproved group(group SU).Conclusions:The cervical spinal canal becomes narrower in CMI patients with an improved syrinx after PFD,indicating a remodeling of the cervical spinal canal.Furthermore,this remodeling process induces milder taper ratios of the cervical spinal canal.更多还原