机械牵张应力对骨化颈椎后纵韧带成纤维细胞的影响

目的探讨机械牵张应力对体外培养的颈椎后纵韧带骨化症（OPLL）患者颈椎韧带成纤维细胞的影响。方法对2012年1月至2013年12月 OPLL 与颈椎外伤但无后纵韧带骨化（非OPLL）患者（各15例）行前路颈椎手术治疗,术中取韧带标本。采用组织块培养法进行细胞体外培养,免疫细胞化学及免疫荧光技术检测胞质波形蛋白。采用 Flexercell 4000细胞加载培养系统分别对两组患者第3代细胞进行机械牵张应力加载,逆转录-聚合酶链式反应（RT-PCR）方法检测两组成纤维细胞应力刺激前及刺激后12、24 h 成骨特异性指标骨钙素、碱性磷酸酶与Ⅰ型胶原 mRNA 表达。结果免疫细胞化学及免疫荧光检测显示胞质波形蛋白呈阳性表达。OPLL组后纵韧带成纤维细胞经机械牵张应力刺激12 h后,骨钙素、碱性磷酸酶及Ⅰ型胶原 mRNA 表达明显升高,应力刺激前后差异具有统计学意义;而非OPLL组应力刺激前后骨钙素、碱性磷酸酶及Ⅰ型胶原 mRNA 表达无明显变化。结论机械牵张应力可促使OPLL患者后纵韧带成纤维细胞骨钙素、碱性磷酸酶及Ⅰ型胶原mRNA表达增加,促进其骨化,其在OPLL进展中发挥重要作用。     

Mechanical strain induces Cx43 expression in spinal ligament fibroblasts derived from patients presenting ossification of the posterior longitudinal ligament

Ossification of the posterior longitudinal ligament (OPLL) is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that mechanical strain can lead to the development of OPLL, although the signaling mechanism is not fully understood. Connexin43 (Cx43), a gap-junction protein, has been shown to be of particular importance in bone formation. We hypothesized that Cx43 may play an important role in the signal transmission induced by mechanical strain during the development of OPLL. To explore this possibility, we cultured fibroblasts from spinal ligaments of OPLL and non-OPLL patients and preloaded mechanical stretch onto the cells via a Flexercell 4000 Tension Plus system. We evaluated expression changes in osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (COL I) and Cx43 via semi-quantitative RT-PCR and western blotting at 12 and 24 h after mechanical strain application in contrast to static conditions. We observed a significant gene up-regulation of OCN, ALP and COL I and Cx43 protein in OPLL cells after mechanical strain application, but no changes in non-OPLL cells. Notably, after RNA interference targeting Cx43 was performed in OPLL cells, we found that there were no significant changes in the expressions of OCN, ALP, COL I and Cx43 after the mechanical strain was applied for 24 h. Thus, we propose that the increase in Cx43 expression induced by mechanical strain in OPLL cells plays an important role in the progression of OPLL.     

颈椎后纵韧带骨化患者韧带成纤维细胞中Cx43的表达及其在成骨化中的作用

目的:观察Connexin43(Cx43)在颈椎后纵韧带骨化患者韧带成纤维细胞中的表达,探讨其在成骨化过程中的作用.方法:选择2013年1月～12月期间在我科行颈前路手术治疗的15例颈椎后纵韧带骨化症患者(骨化组)与15例不伴后纵韧带骨化的颈椎疾病患者(非骨化组).术中切取两组患者韧带标本并采用组织块培养法进行细胞体外培养,应用免疫细胞化学及免疫荧光技术检测胞浆内波形蛋白进行细胞鉴定.采用RT-PCR方法检测细胞内成骨特异指标骨钙素(OCN)、碱性磷酸酶(ALP)及Ⅰ型胶原(COL Ⅰ)mRNA表达,Western blot技术检测两组细胞Cx43蛋白表达,评估其成骨活性.采用siRNA技术对骨化组第3代细胞之Cx43抑制72h,检测抑制组与非抑制组OCN、ALP及COL Ⅰ的表达变化.结果:经鉴定培养出的细胞为成纤维细胞.韧带骨化组细胞OCN、ALP与COL Ⅰ的mRNA表达量分别为1.36±0.21、0.53±0.18、1.64±0.37,明显高于非骨化组(0.78±0.21、0.29±0.13、1.01±0.26);骨化组Cx43蛋白表达量(1.00±0.30)亦明显高于非骨化组(0.48±0.18).对骨化组细胞Cx43抑制72h后,其蛋白表达下调69％(P＜0.01);非抑制组OCN、ALP及COL Ⅰ的mRNA表达量为1.54±0.33、1.32±0.36、1.86±0.44,抑制组分别为0.91±0.32、0.47±0.21、0.88±0.29,两组比较差异具有统计学意义(P＜0.05).结论:颈椎后纵韧带骨化症患者韧带成纤维细胞中存在较高水平的Cx43,并在其成骨化过程中发挥着积极作用.     

颈椎后纵韧带骨化成纤维细胞的培养及其生物学特性

目的探讨颈椎后纵韧带骨化(ossification of posterior longitudinal ligament,OPLL)成纤维细胞培养的方法及其生物学特性,为研究OPLL的病理、生理及发生机制等提供生物学基础。方法采集OPLL患者减压过程中未骨化的部分和颈椎外伤手术减压过程中正常后纵韧带,用原代培养的方法进行体外培养,倒置显微镜观察细胞形态和分泌形成钙结节的过程,并应用Von Kossa方法染色;MTT比色法分析细胞增殖特性;碱性磷酸酶(ALP)活性定量测定和骨钙素(OC)测定比较病变组和正常组差别。结果经Von Kossa染色后镜下显示黑色结节,证实为钙结节;细胞数量从第3 d开始明显增加,并快于正常组;病变组ALP分泌量为(28.56±0.65)U/gprot、OC分泌量为(1.04±0.14)ng/ml,明显高于正常组(P相似文献   

应力对颈椎后纵韧带骨化患者颈椎后纵韧带成纤维细胞蛋白表达的影响

目的:寻找应力刺激前后颈椎后纵韧带骨化(OPLL)患者颈椎后纵韧带成纤维细胞表达差异的蛋白,探讨OPLL的发生机制。方法:从3例行颈前路减压融合内固定术的OPLL患者术中切取后纵韧带组织,清除硬化骨组织后采用组织块培养法培养成纤维细胞,采用机械应力装置对接种于Flexercell板的细胞施加10%、0.5Hz的机械应力,持续加载24h,以同样接种于Flexercell板未施加机械应力的细胞为对照组。提取细胞的总蛋白,采用双向差异凝胶电泳(DIGE)及MALDI-TOF/TOF质谱技术筛选并鉴定表达差异的蛋白,查阅相关文献资料,分析了解其功能特点及其在OPLL发展进程中的可能作用。结果:经过筛选和质谱分析共获得15个具有统计学意义的表达差异蛋白点,其中14个在应力刺激后的后纵韧带细胞中表达下调,1个表达上调。主要是代谢酶类、翻译相关蛋白、细胞骨架蛋白等相关的蛋白,查阅相关文献资料后提示4个表达差异的蛋白与骨化密切相关。结论:机械牵张应力可诱导颈椎后纵韧带成纤维细胞多个与骨化相关的蛋白发生差异性表达,其可能与OPLL发生有关。     

Response of peripheral lymphocytes from patients with ossification of posterior longitudinal ligament

The in vitro response of peripheral blood mononuclear cells or enriched CD4+ T cells from patients with ossification of the posterior longitudinal ligament to anti-CD3 monoclonal antibody has been studied. The response in both was significantly lower in patients with the continuous-type ossification than in patients with the segmental-type ossification and in healthy volunteers, and was inversely correlated with the number of vertebral bodies with ossified ligament. In patients with the segmental-type ossification, the response of peripheral blood mononuclear cells was significantly lower than that in healthy volunteers, but that of the enriched T cells was not. B cell proliferation in response to fixed Staphylococcus aureus cells was significantly lower in patients with the continuous-type ossification than in healthy volunteers but was not correlated with the number of vertebral bodies with ossified ligament. The B cell response in patients with the segmental-type ossification was not lower than that in healthy volunteers. Serum concentrations of transforming growth factor-beta1 and basic fibroblast growth factor also were higher in patients with the continuous-type ossification than in patients with the segmental-type ossification and in healthy volunteers. The findings raise the possibility that continuous-type ossification of posterior longitudinal ligament might develop differently from segmental-type ossification.     

Metabolite profiling of plasma in patients with ossification of the posterior longitudinal ligament

Background

Metabolomics is one of the “omics” technologies, and is a comprehensive analysis of small molecule metabolites which include amino acid, nucleotides, carbohydrates and fatty acid. The purpose of the present study was to compare the differences of metabolite profiling between patients with ossification of the posterior longitudinal ligament (OPLL) and control subjects.

Quality of life in elderly patients with ossification of the posterior longitudinal ligament

STUDY DESIGN: A longitudinal cohort study of 216 elderly patients with ossification of the posterior longitudinal ligament for an average of 13 years was performed. OBJECTIVE: To know the quality of life experienced by patients after treatment. SUMMARY OF BACKGROUND DATA: No report is available on the quality of life experienced by elderly patients with ossification of the posterior longitudinal ligament. Because the life prognosis of patients with this condition is relatively good, the quality of life experienced by elderly patients with this disease is an important subject. METHODS: The study participants were 216 elderly patients with ossification of the posterior longitudinal ligament. Conservative therapy was performed for 126 patients, and surgical therapy for 90 patients. Surgery was basically indicated for patients with myelopathy, who were classified using Nurick's grading system. The cumulative survival rate of these patients and their disabilities in daily living were reviewed. The occurrence of fracture resulting from osteoporosis was surveyed, and the relation of such fractures to bone mineral density was examined. RESULTS: The cumulative survival rate of 70-year-old patients exhibiting Nurick Grade 5 severe myelopathy before treatment was 20%, whereas that of patients without myelopathy or those with Grades 1, 2, 3, or 4 myelopathy before treatment was 80%. Patients who underwent surgical therapy for Grade 3 or 4 myelopathy were statistically more likely to be independent of assistance with activities of daily living than those with similar degrees of myelopathy who underwent conservative therapy. The final quality of life was poor for patients with Grade 5 myelopathy at the first examination, regardless of therapeutic method. The prevalence of complication by fracture in patients with ossification of the posterior longitudinal ligament was 1.4% for men and 8.6% for women. The bone mineral density in these patients without myelopathy was significantly higher than in healthy subjects of the same age. CONCLUSION: The study data suggest that surgical treatment should be chosen for patients exhibiting moderate myelopathy to obtain satisfactory quality of life for them over a long period.     

Cervical myelopathy caused by ossification of the posterior longitudinal ligament

The clinical material presented by the authors includes 24 observations of patients with ossification of the posterior longitudinal ligament. In all cases there was a cervical spine injury in the patients', past history. The diagnosis was confirmed roentgenologically and by the method of computer tomography. The treatment is surgical and consists in anterior decompression of the spinal cord. Recovery was observed in 10 patients (42 +/- 10%), partial regress of myelopathy in 9 patients (29 +/- 10%) and discontinuation (12 +/- 6%); there was no effect in 2 patients of progress of the disease in 3 patients (8 +/- 6%) patients. The authors consider it is necessary to make a careful examination of the patients with myelopathy having a cervical spine injury in their past history in order to reveal ossification of the posterior longitudinal ligament and carry on timely surgical treatment.     

Cervical cord compression from ossification of the posterior longitudinal ligament in non-orientals

We report 14 cases of symptomatic ossification of the posterior longitudinal ligament (OPLL) diagnosed in non-oriental men between 1978 and 1985. All 14 patients had incomplete spinal cord syndromes due to OPLL in the cervical spine and had been referred undiagnosed from other institutions. Twelve had severe myelopathy and seven were wheelchair-bound before OPLL was diagnosed, while six patients had had operations elsewhere for their neurological dysfunction. There was a close association between OPLL and diffuse idiopathic skeletal hyperostosis (Forestier's disease) on plain radiographs, seven patients having both disorders. Enhanced CT scans proved to be the best diagnostic method for the localisation of cord compression, and magnetic resonance imaging, used on four recent cases, provided the best visualisation of the extent of involvement in the sagittal plane. We aim to heighten awareness of OPLL in non-orientals, in whom the clinical features, histological characteristics, and radiographic patterns are very similar to those of oriental patients.     

Operative results and postoperative progression of ossification among patients with ossification of cervical posterior longitudinal ligament

Although the pathogenesis of ossification of the cervical posterior longitudinal ligament (OPLL) has not yet been clarified, it has come to be widely recognized that severe cervical myelopathy or radiculopathy is caused by OPLL. Fifty-three cases who were operated on for OPLL with myelopathy or radiculopathy in our clinic over the past 16 years were followed up. A recovery rate of approximately 70% was observed. Postoperative progressions of the ossification were observed among 75% of the cases of continuous and mixed type but seldom among those with segmental and other types. As causative factors for these postoperative progressions of the ossification, the authors would like to advocate biological, structural, and mobility-related elements. We concluded that in the ossified stage it is desirable to apply anterior decompression for the segmental and other type, posterior decompression for the continuous and mixed type, and, if necessary, two-stage combined decompression for the mixed type.     

Bone mineral analysis in patients with ossification of the posterior longitudinal ligament of the spine

The bone mineral content of the radial metaphysis, the radial diaphysis, the third lumbar vertebral bone and the second metacarpal bone were measured in 78 patients with ossification of the posterior longitudinal ligament (OPLL), and the results were compared with those of 170 age-matched controls. The following results were obtained. Bone mineral content was decreased in cases scoring less than 12 points in the Japanese Orthopaedic Association (JOA) myelopathy score. In males, the mild myelopathy group showed a significantly higher bone mineral content than the control group. In females, however, there was no significant difference between the mild myelopathy and control groups. This may be explained by the marked influence of age on the bone mineral content of females. These results suggest that mineral content in patients with OPLL has a tendency to increase, while the bone mineral content decreases in the severe myelopathy group.     

脊柱后纵韧带骨化性疾病的基础研究进展

脊柱后纵韧带骨化（ossification of posterior longitudinal ligament,OPLL）是指脊柱后纵韧带发生病理性异位骨化,多见于颈椎,常独立或合并其他病变对脊髓神经根形成压迫。在年龄≥65岁的亚洲人群中发病率可达20％～34％。日本学者对该病研究较多,目前的研究表明OPLL是一种基于多基因与环境等其他因素相关的复杂疾病,具体发病机制不明。近年来,对OPLL的基础研究随着生物科学技术的发展取得了一些进展,本文就OPLL的基础研究进展作如下综述。     

颈椎后纵韧带骨化症合并硬膜囊骨化的前路手术治疗

目的 探讨颈椎后纵韧带骨化症合并硬膜囊骨化的影像学表现、前路手术方法 及疗效.方法 2005年1月至2008年3月,前路手术治疗颈椎后纵韧带骨化症合并硬膜囊骨化患者13例.男11例,女2例;年龄43～72岁,平均53.6岁.骨化物分型:局限型3例,分节型2例,连续型5例,混合型3例;骨化物范围涉及1～5椎,平均2.8椎.患者均通过前路椎体次全切除术,切除骨化后纵韧带减压,术中6例患者后纵韧带骨化和硬膜囊骨化得以完全分离,硬膜囊保留完整,另7例患者硬膜囊出现不同程度撕裂或缺损.结果 8例患者术前CT横断面成像上表现为典型的"双影征",2例患者表现为整块骨化物存在中心低密度影,余3例患者表现为椎管狭窄率超过90%的严重后纵韧带骨化.术后5例患者并发脑脊液漏,其中3例经卧床休息、局部加压治疗3～5 d后愈合,另2例患者皮肤愈合后形成间歇性脑脊液囊肿,经反复穿刺抽液治疗1个月后痊愈.随访6个月～2年,平均1年,所有患者JOA评分从术前平均8.1分提高至术后平均13.2分,神经功能恢复率平均57.3%.骨化硬膜囊切除和未切除两组患者的神经功能恢复率差异无统计学意义.结论 CT三维重建检查有助于术前诊断后纵韧带骨化合并硬膜囊骨化,合并硬膜囊骨化并非前路手术的禁忌证,前路手术切除骨化后纵韧带、彻底减压是提高此类患者手术疗效的关键.     

Computer-assisted measurement of the size of ossification in patients with ossification of the posterior longitudinal ligament in the cervical spine

Background Progression of ossification of the posterior longitudinal ligament in patients may lead to serious neurological deterioration. A government-funded study group established a manual method of measurement on plain radiographs to detect progression of the ossified lesion. However, this method did not gain wide acceptance because it was time-consuming and complicated, for which drawings of many lines and points are required. We have applied a computer-assisted measurement system to this task and have evaluated inter- and intraexaminer reliability, showing that it is quicker to use and more accurate than the manual method. Methods Eight board-certified spine surgeons, acting as the examiners, measured the sizes of the ossified lesions on nine lateral cervical spine radiographs using the computer-assisted measurement system. Following insertion of digitized radiographic image data into a computer, the corners of the vertebral bodies on the displayed images are marked by the examiners, and the software automatically sets reference lines and points. The examiners identify upper, lower, and posterior margins of the ossified lesions, and the software calculates the dimensions of the ossified lesions. Data obtained from eight examiners for length and thickness underwent rigorous statistical analysis by calculating the intraclass correlation coefficients with 95% confidence intervals (CIs) to determine interexaminer reliability and Pearson's correlation coefficients between the two measurements by the same examiner to determine intraexaminer reliability. Results The intraclass correlation coefficients were 0.927 and 0.968 with 95% CIs of 0.883–0.955 and 0.956–0.978 for measurements of length and thickness, respectively, of the ossified lesions. The Pearson's correlation coefficients for the two measurements by the same examiners were 0.943–0.985 for length and 0.957–0.991 for thickness. Conclusions The inter- and intraexaminer reliability using this measurement system was excellent. The method can detect progression of ossification of the posterior longitudinal ligament (OPLL) on plain radiographs with high precision and could become a standard method for measuring the size of OPLL.     

颈椎后纵韧带骨化术后C5神经根麻痹

目的探讨颈椎后纵韧带骨化患者后路减压术后发生C5神经根麻痹的临床特点、病理机制及其危险因素。方法2000年1月至2005年8月，采用后路椎板切除减压及侧块钉棒（板）系统固定治疗颈椎后纵韧带骨化患者49例，男35例，女14例；年龄39-75岁，平均53．7岁。在术后6-64h，9例患者发生C5神经根麻痹，表现为患肢三角肌和（或）肱二头肌肌力下降，伴有肩部及上臂外侧感觉减退或消失。比较麻痹患者与非麻痹患者侧位X线片上颈椎前凸角度的变化、C4-5水平CT横断面上椎管狭窄率和MRI T2加权像脊髓高信号区改变的差异。结果9例C5神经根麻痹患者均接受保守治疗，治疗措施包括功能锻炼、口服药物及高压氧治疗。随访1-4年，平均2．2年，9例患者肌力均恢复至3-4级。麻痹患者颈椎曲度矫正值（12．5°±3．0°）大于非麻痹患者（1．9°±1．1°），差异有统计学意义（P=0．04）；麻痹患者椎管狭窄率（62．6％±6．8％）高于非麻痹患者（35．1％±5．4％），但差异无统计学意义（P=0．12）；麻痹患者术后仅有3例脊髓高信号区范围扩大而超过原椎间隙水平，与非麻痹患者脊髓高信号区改变的比例差异无统计学意义（P=0．32）。结论C5神经根麻痹在颈椎后纵韧带骨化患者中具有较高发生率，后路手术中应适度矫正颈椎前凸角度。