胸椎黄韧带骨化症与骨形态发生蛋白4基因单核苷酸多态性的关联

背景：骨形态发生蛋白4基因与胸椎黄韧带骨化症的相关性研究目前较少。 目的：测观察骨形态发生蛋白4的2个单核苷酸多态性位点rs17563和rs2855532与胸椎黄韧带骨化症的关联。 方法：胸椎黄韧带骨化症患者和正常对照者各40例,收集受试者的周围静脉血提取DNA,PCR法进行目的片段骨形态发生蛋白4的单核酸多态性位点rs17563和rs2855532的扩增并测序。 结果与结论：黄韧带骨化症组中rs17563和rs2855532位点带“T”基因型及等位基因型频率明显高于对照组(P < 0.05)。证实,骨形态发生蛋白4上的2个单核酸多态性位点rs17563和rs2855532等位基因型突变与胸椎黄韧带骨化症的发生有关。     

胸椎椎板倾斜角在胸椎黄韧带骨化中的解剖学意义

目的:探讨胸椎椎板倾斜角在胸椎黄韧带骨化中的解剖学意义。方法:(1)正常干燥胸椎标本(T1￣T12)20具,在棘突与椎板交界处用量角器测量椎板后表面与椎体水平面的夹角即椎板倾斜角,分析其分布规律;(2)计数22例胸椎黄韧带骨化症患者的骨化节段数,观察其分布情况。比较两者分布情况。(3)将黄韧带与相邻椎板视为“椎板-黄韧带-椎板复合体”,分析黄韧带在不同椎板倾斜角时受力。结果:胸椎椎板倾斜角以T7￣T10为波谷段,胸椎黄韧带骨化以T8￣T10为波峰段,两者分布具有相关性,椎板倾斜角最小的下胸段与黄韧带骨化好发部位一致。黄韧带受轴向牵拉力为F·sinα,在椎板垂直的理想状态下黄韧带受力为F,α越大,黄韧带受到的张力越小。结论:胸椎椎板倾斜角和黄韧带所受张力可能是胸椎黄韧带骨化多发于下胸段的解剖学和力学因素之一。     

人黄韧带细胞骨化发生过程中的自噬

文题释义： 黄韧带骨化症：是由于脊柱黄韧带发生骨向分化而形成的一种韧带骨化疾病,常导致椎管狭窄,造成脊髓受压,并产生不可逆损害,出现肢体感觉障碍,严重的将导致瘫痪。目前对黄韧带骨化病因学研究取得了一定的进展,但其具体的发病机制仍未完全明确。 自噬：因外界或自身环境的改变,细胞器和胞内蛋白通过溶酶体进行分解消化并重新利用,从而达到适应外界环境、维持细胞内环境稳定的过程,是真核细胞特有一种表现形式。 MAPK信号通路：是生物体内重要的信号转导系统之一,它可以被物理应激、炎性细胞因子、生长因子以及细胞复合物等一系列细胞外信号或刺激激活,从而参与介导细胞生长、发育、分裂以及分化等多种生理及病理过程。在哺乳动物细胞中MAPK信号通路的主要信号分子包括ERK1/2、JNK以及P38等。 背景：黄韧带骨化发生的病理机制尚不清楚,临床上无有效的药物或非手术治疗的方法。目前研究发现,骨桥蛋白与自噬在成骨过程中均发挥重要作用,二者在黄韧带骨化中的作用尚不清楚。 目的：通过对骨桥蛋白和自噬在黄韧带骨化发生机制的研究,尝试找出药物治疗的潜在作用靶点。 方法：①黄韧带骨化病、胸椎骨折或单纯腰椎间盘突出症患者行后路全椎板切除减压获取黄韧带,将标本分为骨化组和非骨化组,每组各取8例标本,通过免疫组化染色观察骨桥蛋白、骨钙素及自噬指标Beclin-1、LC3、P62的表达;②通过组织块贴壁法进行黄韧带细胞的分离培养,并用不同质量浓度的骨桥蛋白干预不同时间来构建体外黄韧带细胞骨化模型;③非骨化组黄韧带细胞用不同浓度的自噬抑制剂3-甲基腺嘌呤干预后,再用100 μg/L骨桥蛋白诱导,应用Western blot检测骨化指标碱性磷酸酶、骨钙素的表达变化;④用100 μg/L骨桥蛋白干预非骨化黄韧带细胞,并在0,15,30,60,120 min终止骨化诱导过程,应用Western blot检测MAPK信号通路中重要分子ERK1/2、JNK、P38的磷酸化情况;⑤非骨化组黄韧带细胞用ERK1/2特异的磷酸化阻滞剂U0126阻断ERK1/2通路磷酸化后,再用100 μg/L骨桥蛋白诱导,应用Western blot检测碱性磷酸酶以及骨钙素的表达变化。 结果与结论：①骨化黄韧带和非骨化黄韧带组织骨钙素、骨桥蛋白的免疫组化均呈阳性表达;骨化黄韧带组织中Beclin-1呈阳性表达,而LC3及P62未见明显阳性结果;非黄韧带骨化组织中Beclin-1、LC3、P62均呈阳性表达;②与非骨化组比较,骨化组黄韧带细胞中碱性磷酸酶和骨钙素的表达增加;骨桥蛋白可诱导黄韧带骨化,骨桥蛋白的作用具有浓度相关性和时间相关性;③自噬强弱与骨化程度呈负相关,即自噬越明显,骨化作用越弱;④骨桥蛋白能使MAPK信号通路磷酸化,并具有一定的时间相关性;抑制MAPK磷酸化过程后,骨桥蛋白仍然能够诱导黄韧带细胞的骨化;⑤结果表明,黄韧带骨化发生过程中,信号通路上ERK1/2、骨桥蛋白、骨钙素、碱性磷酸酶分子的上下游关系为：ERK1/2→骨桥蛋白→骨钙素/碱性磷酸酶。 ORCID: 0000-0003-0008-9802(许国峰) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

胸椎黄韧带骨化的影像学研究及其临床意义

目的:探讨胸椎黄韧带骨化的影像学表现,评价X线、CT和MRI的诊断价值.方法:回顾性分析23例经手术病理证实的胸椎黄韧带骨化的X线、CT和MRI影像学资料.结果:所有病例的X线平片显示胸椎退行性改变,仅其中3例的侧位片显示椎间孔处模糊骨化影.CT扫描显示胸椎椎板前缘凸向椎管的高密度影,椎管变窄,外侧型7例,弥漫型8例,结节增厚型8例.MRI显永黄韧带骨化病变呈低信号影,脊髓不同程度受压.23例患者共发现黄韧带骨化节段57个,骨化节段为单发病灶6例,多发病灶17例.病变累及上胸段(T1-4)3例,中胸段(5-8)7例,下胸段(T9-12)16例.合并后纵韧带骨化6例,合并胸椎间盘突出2例.结论:胸椎黄韧带骨化有典型的CT和MRI影像学表现,但在X线平片上容易漏诊;CT结合MRI检查是确诊胸椎黄韧带骨化的蕈要诊断手段.     

手术治疗腰椎黄韧带骨化症11例分析

<正>黄韧带骨化症(ossification of ligamentum flavum,OLF)为老年性疾病,多见于日本及东南亚人群,欧美等非亚洲人群仅见个案报道。黄韧带骨化可发生于脊柱各部,以胸椎及胸腰椎多见,颈椎及下腰椎少见。腰椎黄韧带骨化发生率较低,此类临床报     

胸椎黄韧带骨化手术前后的护理

胸椎黄韧带骨化因其诊断困难,常被漏诊和误诊,但近年来已被人们逐渐认识并引起重视。该病主要累及椎板间连接组织——黄韧带发生增生肥厚、骨化,最终导致椎体边缘唇样增生,椎间隙变窄,从而引起椎管狭窄和脊髓的压迫性损害。本病多发生于东亚地区,日本报道较早,国内胸椎黄韧骨化仅有少数病例报告。本文总结我所1983～1994年手术治疗48例胸     

尸体胸椎黄韧带骨化的病理观察

研究兴柱黄韧带骨化的病理特征。方法：随机选择２１具尸体胸椎标本，矢状剖开１８具，冠状剖开３具，观察每一节段黄韧带病理学特征，对骨化标本进行病理学研究。结果；２１具标本中，９具５９节段骨化，其中增生骨化４具，２３节段占３９％，单纯骨化７具，累积３６节段，病理表现呈３层结构。     

脊柱黄韧带骨化中血管生成的作用和临床意义

目的探讨血管生成在脊柱黄韧带骨化中的作用和临床意义。方法3例胸腰椎骨折,10例退变性腰椎管狭窄和14例胸椎黄韧带骨化的后路手术黄韧带标本,分别作为正常组(N)、退变组(D)和骨化组(O),均做HE染色和血管内皮生长因子(VEGF)、环氧化酶2(COX2)和CD34免疫组化染色,测定微血管密度(MVD),BI2000定量分析VEGF、COX2积分光密度值(IOD)。结果正常组纤维母细胞VEGF、COX2表达阴性,退变和骨化组纤维母细胞增生并表达阳性,两组IOD值t检验,骨化组阳性率高于退变组。微血管在正常组稀少,在韧带与骨化移行区增生明显,可见大量CD34阳性表达的血管内皮前体细胞。3组MVD值q检验,骨化和退变组阳性率高于正常组,骨化和退变组无差异。微血管内皮细胞间距增大,管壁缺乏基膜和平滑肌层,管周见阴性染色的细胞聚集。结论脊柱黄韧带纤维母细胞异常分泌VEGF和COX2,促进局部血管新生,是骨化必备条件。     

环抱法切除胸椎黄韧带骨化症的临床疗效与解剖学基础

目的 明确I期后入路徒手环抱法切除胸椎黄韧带骨化症的手术指征、临床疗效与解剖学基础。方法 回顾2010年1月至2018年6月符合以下指征行I期后入路徒手环抱法手术切除胸椎骨化黄韧带患者20例（男9例,女11例,平均年龄55.2岁,43至75岁）：第一诊断为胸椎黄韧带骨化;排除Sato分型中的融合型及结节型;排除术前CT检查具有硬膜骨化征患者;排除各节段的单侧骨化物椎管占有率大于55%患者。收集术前与末次随访的改良JOA评分、临床解剖学特点及手术时间、出血量、并发症等临床资料,予以分析总结。结果 20例患者均行环抱法经后路I期切除骨化韧带,共36个节段。平均手术时间116 min,出血量131 mL。术中有1例脑脊液漏,术中予缝合,术后无症状。病例均得以随访,随访时间48～54个月,平均50.9个月。术前mJOA评分（7.50±1.02）,末次随访（10.25±0.62）,差异有统计学意义（P<0.05）。术后改善率平均80.43%,优13例,良7例。未出现复发病例。结论 对符合手术指征的黄韧带骨化患者行环抱法切除术具有操作简单,辅助工具少,手术时间短,效疗确切的优点。术后未见复发...     

三维可视化技术在胸椎黄韧带骨化症手术治疗中的应用

目的 探讨三维可视化技术辅助设计手术治疗胸椎黄韧带骨化症的临床应用价值。方法 回顾性分析2013年11月—2016年12月郑州市骨科医院脊柱一科采用椎板切除治疗的44例胸椎黄韧带骨化症的临床资料。其中,男25例、女19例,年龄40～76岁。术前均采用Mimics软件对胸椎CT扫描的DICOM数据进行三维重建,在胸椎椎板和骨化黄韧带的三维可视化模型上观察骨化灶的立体结构以及与椎弓根、关节突、椎板的位置关系,计算数字模拟骨化灶体积;按照“分区椎板切除”的方式在三维可视化数字模型上进行骨化黄韧带的模拟切除;按照术前模拟切除步骤,实施手术操作。观察手术时间、术中出血量及并发症发生情况。术后12个月采用日本骨科协会(JOA)评分评估神经功能,根据术前和术后JOA评分计算JOA改良率,评估手术疗效。结果 三维重建模型44个,重建骨化灶103个,骨化灶体积为(1 831±443)mm³。44例患者均顺利完成手术,手术时间65～180(96.7±19.6)min;术中出血量230～1 350(432±83.5)mL。患者术后无脊髓神经损害症状加重者,术后神经症状均逐步好转。44例患者均获随访,随访时间13～46(25±10.3)个月。随访期间无迟发性感染、神经症状加重、内固定失败等并发症发生。术后12个月JOA评分(8.8±1.8)分,明显高于术前的(5.3±2.0)分,差异有统计学意义(t=11.566, P＜0.01);JOA评分改善率为64.2%±21.7%,疗效评价优13例、良21例、可10例,优良率77.3%(34/44)。结论 应用三维可视化技术进行术前评估,能够立体、全面了解骨化黄韧带的形态,通过模拟手术,设计手术切除范围,可以提高手术精准度、安全性和有效性,为胸椎黄韧带骨化症的诊疗提供了一种新的术前影像学辅助方法。     

Tissue transglutaminase is involved in mechanical load–induced osteogenic differentiation of human ligamentum flavum cells

Mechanical load–induced osteogenic differentiation might be the key cellular event in the calcification and ossification of ligamentum flavum. The aim of this study was to investigate the influence of tissue transglutaminase (TGM2) on mechanical load–induced osteogenesis of ligamentum flavum cells. Human ligamentum flavum cells were obtained from 12 patients undergoing lumbar spine surgery. Osteogenic phenotypes of ligamentum flavum cells, such as alkaline phosphatase (ALP), Alizarin red-S stain, and gene expression of osteogenic makers were evaluated following the administration of mechanical load and BMP-2 treatment. The expression of TGM2 was evaluated by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA) analysis. Our results showed that mechanical load in combination with BMP-2 enhanced calcium deposition and ALP activity. Mechanical load significantly increased ALP and OC gene expression on day 3, whereas BMP-2 significantly increased ALP, OPN, and Runx2 on day 7. Mechanical load significantly induced TGM2 gene expression and enzyme activity in human ligamentum flavum cells. Exogenous TGM2 increased ALP and OC gene expression; while, inhibited TG activity significantly attenuated mechanical load–induced and TGM2-induced ALP activity. In summary, mechanical load–induced TGM2 expression and enzyme activity is involved in the progression of the calcification of ligamentum flavum.     

Involvement of p38MAPK/NF-κB Signaling Pathways in Osteoblasts Differentiation in Response to Mechanical Stretch

Bone morphogenetic proteins (BMPs) are known to be important in osteoblasts' response to mechanical stimuli. BMPs/Smad signaling pathway has been demonstrated to play a regulatory role in the mechanical signal transduction in osteoblasts. However, little is currently known about the Smad independent pathway in osteoblasts differentiation in mechanical loading. In this study, MC3T3-E1 cells were subjected to mechanical stretch of 2000?micro-stain (με) at 0.5?Hz, in order to investigate the involvement of p38MAPK and NF-κB signaling pathways in mechanical response in osteoblasts. We found BMP-2/BMP-4 were up-regulated by mechanical stretch via the earlier activation of p38MAPK and NF-κB signaling pathways, which enhanced osteogenic gene expressions including alkaline phosphatase (ALP), collagen type I (Col I) and osteocalcin (OCN), and the expressions of these osteogenic genes were remarkably decreased with Noggin (an inhibitor for BMPs signals) pretreatment. Furthermore, BMP-2/BMP-4 expressions were suppressed by PDTC, an inhibitor of NF-κB pathway and SB203580, an inhibitor of p38MAPK pathway, respectively, leading to the declined levels of ALP, Col I and OCN. Interestingly, blocking in p38MAPK pathway can also cause the inactivation of NF-κB pathway in mechanical stretch. Collectively, the results indicate during mechanical stretch p38MAPK and NF-κB signaling pathways are activated first, and then up-regulate BMP-2/BMP-4 to enhance osteogenic gene expressions. Moreover, p38MAPK and NF-κB signals have cross-talk in regulation of BMP-2/BMP-4 in mechanical response.     

动静态不同牵张条件下大鼠骨髓间充质干细胞的增殖与分化

背景：在体外和体内关于细胞对于不同的机械牵张反应的大量研究表明,牵张能够刺激成骨。然而鲜有文献报道不同的牵张方式对于同种细胞的影响有何不同。 目的：比较不同机械牵张方式对大鼠骨髓间充质干细胞的影响。 方法：分离培养大鼠骨髓间充质干细胞,应用自行研制的牵张装置对骨髓间充质干细胞分别施加动态、静态和模拟临床的混合牵张牵张刺激,分别检测3种刺激方式下骨髓间充质干细胞的增殖能力、碱性磷酸酶活性及Runx2基因的mRNA表达,并测量细胞骨钙素的分泌情况。 结果与结论：静态牵张组与对照组相比,细胞增殖能力提高18.67%,碱性磷酸酶活性、Runx2表达及骨钙素分泌无明显差异;动态牵张组相对于对照组,细胞碱性磷酸酶活性提高60.33%, Runx2表达上升49.67%,细胞外骨钙素的分泌提高了48%,然而细胞增殖则受到了抑制;混合牵张组相对于对照组,细胞增殖能力稍有上升但无统计学差异,其对碱性磷酸酶活性、Runx2表达以及骨钙素的分泌有一定的促进作用,但没有动态牵张组明显。结果提示,静态牵张能够显著刺激骨髓间充质干细胞的增殖,而动态牵张对于刺激骨髓间充质干细胞成骨向分化作用更为明显,混合牵张方式对于细胞增殖及成骨分化均有一定的促进作用。     

骨桥蛋白及其受体在黄韧带骨化症黄韧带细胞中的表达及其意义

BACKGROUND: The exact pathogenesis of ossification of ligamentum flavum has not been elucidated yet. And osteopontin may be an important factor involved in the ossification of ligamentum flavum. OBJECTIVE: To clarify the expression and significance of osteopontin and its receptors, CD44 and integrin-β3, in ligamentum flavum cells between normal controls and patients with ossification of ligamentum flavum.   METHODS: Ligamentum flavum tissues were obtained from normal adult controls and adult patients with ossification of ligamentum flavum (n=8 per group) who underwent thoracic/lumbar posterior decompression surgery. Ligmentum flavum cells were separated, cultured and identified in vitro, and osteopontin, CD44, integrin-β3 were stained using immunocytochemistry method and observed under inverted phase contrast microscope. And the mRNA expressions of osteopontin, CD44, integrin-β3 were measured by RT-PCR. RESULTS AND CONCLUSION: Immunocytochemistry results showed that the stronger positive staining for osteopontin, CD44, integrin-β3 was observed in the ossification of ligamentum flavum group than the control group (P < 0.01). The mRNA expressions of osteopontin, CD44 and integrin-β3 were also higher in the ossification of ligamentum flavum group than the control group (P < 0.05). These findings indicate that osteopontin and its receptors, CD44 and integrin-β3, in ligamentum flavum cells may play an important role in ossification of ligamentum flavum.      

Osteogenesis with cryopreserved marrow mesenchymal cells

Rat marrow cells were collected from the femurs of 7-week-old male rats (Fischer 344), cultured in 75-cm2 flasks for 10 days, released with trypsin, and then frozen and stored at -196 degrees C in liquid nitrogen. Three months later, the cryopreserved marrow cells were rapidly thawed and cultured in porous hydroxyapatite (HA) blocks in osteogenic medium containing 10 mM sodium beta-glycerophosphate, vitamin C phosphate (82 microg/mL), and 10 nM dexamethasone. After 2 weeks of subculture, cultured cells-HA constructs were subcutaneously implanted into syngeneic rats. The constructs were harvested 2 and 4 weeks postimplantation and examined by histological, biochemical, and genetic analyses. Histological examination showed extensive bone formation in the HA pores. High alkaline phosphatase (ALP) activity and high osteocalcin content were detected in the constructs. Expression of ALP and osteocalcin mRNA was observed at both 2 and 4 weeks. These results indicate that artificial bone prepared with cryopreserved cells had a marked osteogenic capacity.     

骨形态发生蛋白9体外调节乳腺癌细胞与骨髓间充质干细胞的相互作用

 目的 观察骨形态发生蛋白9（BMP9）在共培养条件下对HS-5成骨分化及MDA-MB-231转移相关因子表达的影响。方法 采用Transwell小室间接共培养MDA-MB-231细胞与HS-5细胞,加入BMP9条件培养基,使用化学发光法、细胞化学染色法及茜素红S染色法分别检测HS-5细胞碱性磷酸酶（ALP）活性、ALP表达及细胞钙盐沉积的变化;应用RT-PCR及Western blot观察HS-5细胞骨钙素（OCN）、MDA-MB-231细胞甲状旁腺激素相关蛋白（PTH-rP）及白细胞介素6(IL-6)mRNA和蛋白表达的变化。结果 HS-5的ALP活性升高,并呈时间依赖性,第9天达最高,随后降低;ALP细胞化学染色结果进一步证实这一点;HS-5细胞钙盐沉积增多、OCN mRNA和蛋白的表达升高（P<0.05）;MDA-MB-231转移相关的PTH-rP及IL-6 mRNA和蛋白的表达下降（P<0.05）。结论 BMP9能够调节乳腺癌细胞与骨髓间充质干细胞的相互作用。