椎板切除术后颈椎后凸畸形动物模型构建及持续静态压力下软骨终板细胞凋亡机制的研究
发布时间：2019-06-14 16:14
【摘要】：研究背景 颈椎板切除术已被广泛应用于治疗儿童脊髓损伤、脊髓肿瘤、先天性畸形以及脊髓空洞症等疾病的治疗。然而,椎板切除手术后可能会发生脊柱的后凸畸形并最终导致神经功能障碍,严重影响人类身体健康。因此,深入探讨椎板切术后颈椎后凸畸形的发病机制有着重要科学意义。 在正常情况下,脊柱矢状面的轴向垂线应当通过颈1(C1)和胸1(T1)中心,颈椎前凸使矢状面的轴向垂线在C2-C7的后方。颈椎生理前凸的维持主要依靠颈椎前方、后方骨和软组织结构的完整及力量的均衡。颈椎前方的骨性结构和椎间盘等组织主要承受压力,后方的关节突关节、韧带和肌肉等组织则主要抵抗颈部张力。先前研究表明：椎板切除术后颈椎后凸畸形的发生是由于颈椎后伸力矩的破坏所造成的。颈椎板切除术后颈椎后方的肌肉韧带结构复合体以及骨性结构的完整性遭到了破坏,使得术前后方韧带所承载的牵张力负荷(后伸力矩)丢失,从而引起前方椎体所承载的压力性负荷增加并最终导致畸的形发生。Pal和Sherk等人对颈椎生物力学研究发现64%的轴向性负荷经由两侧的关节突关节均匀承载,颈椎椎板切除术后这个轴向性负荷将向前转移,估计将增加前方椎体10倍于原来的压力性负荷。因青少年椎体生长板正处在生长发育期,椎板切除术后异常压力的增加使得生长板软骨细胞发育迟滞、凋亡加速,出现椎体楔形变从而形成后凸畸形。 软骨终板在维系脊柱的生物力学结构完整性以及椎间盘营养方面起重要作用。近年来,异常应力下生长板软骨细胞凋亡与颈椎后凸畸形的关系逐渐受到重视,对于生长板软骨细胞凋亡是否参与了椎板切除术后颈椎后凸畸形的发生过程目前尚未有研究报道。此外,异常应力通过何种信号通路诱导软骨细胞凋亡亦未被完全阐明。对于这些问题的回答,需要我们做出进一步的深入研究。 目的： 1.建立椎板切除术后青少年颈椎后凸畸形的动物模型,为研究后凸畸形提供有效的研究载体。 2.通过对体外培养的生长板软骨细胞凋亡机制的研究,以期从分子生物学角度解释椎板切除术后青少年颈椎后凸畸形的发病机制。 方法： 1.24只3月龄大山羊随机分成两组：椎板切除术组(n=12),和对照组(n=12)。分别于手术前、手术后当天以及手术后3、6个月在麻醉下拍摄标准颈椎侧位片,并采用后切线夹角法来评估颈椎曲度的变化。通过透射电镜法以及末端脱氧核苷酸转移酶(TdT)介导的d-UTP缺口末端标记技术(TUNEL)来验证软骨终板的细胞凋亡。 2.取体重100-150g SD大鼠的颈椎体生长板软骨进行原代软骨细胞的分离及培养,通过软骨细胞形态以及二型胶原免疫荧光染色等方法鉴定软骨细胞。分别给予第二代软骨细胞施加0、0.1、0.2、0.5MPa持续静态压力,倒置相差显微镜观察软骨细胞形态变化,CCK-8法检测软骨细胞存活率,流式细胞仪以及TUNEL法检测软骨细胞凋亡率,Western blot法检测MAPK以及线粒体凋亡相关蛋白表达情况。 结果： 1.体内实验部分： (1)影像学结果： 术前两组山羊颈椎曲度之间无明显统计学差异,通过术后与术前X光片比较发现三个月后椎板切除手术组山羊开始发生后凸畸形,在第六个月时后凸畸形进一步加重,而对照组山羊颈椎曲度在整个实验时间内并未发生明显变化。术前手术组山羊C2-C5平均后切线夹角为-15.8±0.8。,术后三个月降低为19.1±1.1。而到第六个月的时候则为20.2±0.6。。手术后与手术前后切线夹角的变化揭示了后凸畸形的形成。 (2)软骨细胞凋亡： 在整个观察时间点内对照组生长板仅有少量的凋亡细胞。然而,椎板切除手术组在手术后第三个月即可以观察到大量凋亡的软骨细胞(29.7±3.4%),并且凋亡软骨细胞比例在第六个月时进一步增加(35.6±5.4%),手术组软骨细胞凋亡率与对照组相比差异具有统计学意义。 (3)透射电镜观察： 对照组生长板软骨细胞呈不规则形或椭圆形,细胞膜完整,细胞器丰富,染色质均匀分布。椎板切除术组可见典型的凋亡的软骨细胞,表现为细胞膜皱缩,细胞核碎裂,染色质浓缩以及细胞器的减少。 2.体外实验部分： 1.持续静态压力对终板软骨细胞活力影响： 为研究持续静态压力对生长板软骨细胞存活率的影响,我们分别给予软骨细胞施加不同静态压力(0,0.1,0.2和0.5MPa)加压1,6,12,24,36,和48小时后采用CCK-8法检测细胞存活率。结果显示软骨细胞存活率呈压力以及时间依赖性降低,其中在0.5Mpa大气压下作用24小时细胞存活率约为50%。 2.持续静态压力可以诱导生长板软骨细胞凋亡： 经0.5Mpa大气压加压24小时后,加压组软骨细胞TUNEL的阳性率明显高于对照组,差异具有统计学意义(48.77±4.14%vs.4.43±0.85%,P0.001)。此外我们还采用流式细胞仪AV/PI双染法来验证软骨细胞凋亡,软骨细胞经0.5MPa持续静态压力下作用24小时后,加压组软骨细胞凋亡率较正常组相比有显著性升高(43.79±4.87%vs.5.66±0.97%,P0.001)。 3.持续静态压力经线粒体及Caspase途径诱导软骨细胞凋亡： (1)持续静态压力对软骨细胞线粒体功能以及线粒体凋亡相关蛋白影响； 为研究线粒体功能障碍在持续静态压力诱导生长板软骨细胞凋亡中的作用,我们采用JC-1免疫荧光染色法来检测线粒体膜电位(mitochondrial membrane potential,△Ψm)的改变。经0.5Mpa大气压加压24小时后,加压组软骨细胞红色荧光强度明显弱于未加压组,而绿色荧光强度则明显高于对照组。这些结果提示加压组软骨细胞中线粒体膜电位的降低。此外,实验组软骨细胞经0.5MPa加压24小时后Bax蛋白表达水平较对照组明显升高,而Bcl-2蛋白表达水平则较正常组有显著性的降低。我们进一步检测了从线粒体向细胞浆中释放的细胞色素C的含量,结果显示加压后胞浆中细胞色素C的含量明显增加。 (2)持续静态压力对软骨细胞Caspase蛋白表达影响. 我们采用Western blot方法研究Caspase相关蛋白是否参与了持续静态压力诱导软骨细胞凋亡过程。结果显示,在0.5MPa下加压24小时后实验组较对照组相比活化的Caspase-9以及Caspase-3蛋白均明显升高,差异具有统计学意义。应用Caspase-3抑制剂后活化形式的Caspase-3蛋白表达量明显降低,而且可以显著性的降低持续静态压力诱导的软骨细胞凋亡。 4. MAPK信号通路在持续静态压力诱导的生长板软骨细胞凋亡中的作用： 0.5Mpa持续静态压力刺激生长板软骨细胞24小时后,磷酸化JNK, p38MAPK和ERK1/2蛋白表达水平较未加压组有显著性的升高。与单纯加压组相比,使用10μM SP600125,10μM SB203580和20μM PD98059预处理软骨细胞后再给予持续静态压力可以显著性的降低加压所引起的磷酸化JNK, p38MAPK和ERK1/2蛋白水平的表达。为了进一步研究MAPK在持续静态压力诱导软骨细胞凋亡中的作用,我们加入相应的MAPK抑制剂提1小时处理软骨细胞后再给予持续加压。结果显示应用10μM SP600125,10μM SB203580和20μM PD98059能够显著性的降低持续静态压力诱导的软骨细胞凋亡。此外,提前1小时使用10μM SP600125,10μM SB203580和20μMPD98059预处理软骨细胞可以改善持续静态压力诱导的线粒体膜电位的降低。Western blot结果显示提前1小时使用MAPK相应抑制剂预处理软骨细胞可以上调Bcl-2以及下调Bax蛋白表达水平,并减少细胞色素C从线粒体中的释放。这些结果显示持续静态压力经MAPK-线粒体信号通路诱导终板软骨细胞凋亡。 结论： 1.椎板切除术组生长板软骨细胞凋亡率明显高于对照组,提示生长板软骨细胞凋亡可能在椎板切除术后颈椎后凸畸形的形成过程中起重要作用。 2.持续静态压力可以诱导大鼠颈椎体生长板软骨细胞凋亡。 3.持续静态压力经由线粒体和Caspase途径诱导软骨细胞凋亡。 4. MAPK信号通路参与了持续静态压力诱导的终板软骨细胞凋亡过程。
[Abstract]:Study Background Cervical plate resection has been widely used in the treatment of children's spinal cord injury, spinal cord tumor, congenital malformation, and syringomyelia. Treatment. However, after the laminectomy, the kyphosis of the spinal column may occur and eventually lead to neurological dysfunction, which seriously affects the health of the human body. On the other hand, it is of great significance to study the pathogenesis of the kyphosis of the cervical vertebra after the operation of the disc. Predefined. In normal cases, the axial vertical line of the sagittal plane of the spine should be centered on the neck 1 (C1) and the chest 1 (T1), and the axial vertical of the sagittal plane in the front of the cervical spine is at C2-C7. The anterior and posterior approach of the cervical spine depends on the integrity and strength of the anterior, posterior and soft tissue structures of the cervical spine. The bone structure and the intervertebral disc in the front of the cervical vertebra are mainly subjected to pressure, and the joint of the posterior joint, the ligament and the muscle are mainly against the neck. Department tension. Previous studies have shown that the occurrence of kyphosis after laminectomy is due to the destruction of the cervical posterior extension It is caused by the destruction of the structural complex of the muscle ligament and the integrity of the bony structure of the posterior cervical vertebra after the cervical plate resection, so that the traction tension load (post-extension moment) carried by the anterior posterior ligament ) loss, resulting in an increase in the pressure load carried by the anterior vertebral body and ultimately to a teratogenic The formation of the cervical spine by Pal and Sherk et al. found that 64% of the axial load was uniformly loaded via the articular processes on both sides. After the cervical laminectomy, this axial load will be transferred forward, which will increase the anterior vertebral body by 10 times the original pressure Sexual load. Due to the growth and development stage of the juvenile vertebral body, the increase of abnormal pressure after laminectomy resulted in the development of the growth plate cartilage cell, the acceleration of apoptosis, the formation of a wedge-shaped body of the vertebral body, Convex deformity. The cartilage endplate is used to maintain the biomechanical structural integrity of the spinal column and the aspect of disc nutrition. In recent years, the relationship between the apoptosis of the growth plate and the kyphosis of the cervical vertebra has been paid more and more attention. in addition, what signal path of abnormal stress induce that apoptosis of the chondrocyte is also not It's fully set out. For answers to these questions, we need to make further in-depth Objective:1. To establish an animal model of posterior cervical kyphosis of young people after laminectomy, in order to study kyphosis. To provide an effective study vector.2. The study of the mechanism of apoptosis in the growth plate of the growth plate in vitro, with a view to the interpretation of the young people after laminectomy from the point of molecular biology After cervical vertebra Methods: 1.24 3-month-old goats were randomly divided into two groups: the laminectomy group (n = 12), and the control group (n = 12). TUN-mediated d-UTP nick end-end labeling technique (TUN) mediated by transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase (TdT) EL) to verify the cell apoptosis of the cartilage endplates.2. The separation and culture of primary chondrocytes from 100-150 g SD rats with body weight of 100-150 g SD rats were carried out. Cartilage cells were identified by collagen immunofluorescence staining and the like.0, 0.1, 0.2, 0.5 MPa continuous static pressure were applied to the second generation of chondrocytes, and the morphological changes of the chondrocytes were observed by an inverted phase contrast microscope. The survival rate of the chondrocytes was detected by the CCK-8 method. The flow cytometry And the apoptosis rate of the cartilage cells was detected by the TUNEL method. PK浠，
本文编号：2499516
[Abstract]:Study Background Cervical plate resection has been widely used in the treatment of children's spinal cord injury, spinal cord tumor, congenital malformation, and syringomyelia. Treatment. However, after the laminectomy, the kyphosis of the spinal column may occur and eventually lead to neurological dysfunction, which seriously affects the health of the human body. On the other hand, it is of great significance to study the pathogenesis of the kyphosis of the cervical vertebra after the operation of the disc. Predefined. In normal cases, the axial vertical line of the sagittal plane of the spine should be centered on the neck 1 (C1) and the chest 1 (T1), and the axial vertical of the sagittal plane in the front of the cervical spine is at C2-C7. The anterior and posterior approach of the cervical spine depends on the integrity and strength of the anterior, posterior and soft tissue structures of the cervical spine. The bone structure and the intervertebral disc in the front of the cervical vertebra are mainly subjected to pressure, and the joint of the posterior joint, the ligament and the muscle are mainly against the neck. Department tension. Previous studies have shown that the occurrence of kyphosis after laminectomy is due to the destruction of the cervical posterior extension It is caused by the destruction of the structural complex of the muscle ligament and the integrity of the bony structure of the posterior cervical vertebra after the cervical plate resection, so that the traction tension load (post-extension moment) carried by the anterior posterior ligament ) loss, resulting in an increase in the pressure load carried by the anterior vertebral body and ultimately to a teratogenic The formation of the cervical spine by Pal and Sherk et al. found that 64% of the axial load was uniformly loaded via the articular processes on both sides. After the cervical laminectomy, this axial load will be transferred forward, which will increase the anterior vertebral body by 10 times the original pressure Sexual load. Due to the growth and development stage of the juvenile vertebral body, the increase of abnormal pressure after laminectomy resulted in the development of the growth plate cartilage cell, the acceleration of apoptosis, the formation of a wedge-shaped body of the vertebral body, Convex deformity. The cartilage endplate is used to maintain the biomechanical structural integrity of the spinal column and the aspect of disc nutrition. In recent years, the relationship between the apoptosis of the growth plate and the kyphosis of the cervical vertebra has been paid more and more attention. in addition, what signal path of abnormal stress induce that apoptosis of the chondrocyte is also not It's fully set out. For answers to these questions, we need to make further in-depth Objective:1. To establish an animal model of posterior cervical kyphosis of young people after laminectomy, in order to study kyphosis. To provide an effective study vector.2. The study of the mechanism of apoptosis in the growth plate of the growth plate in vitro, with a view to the interpretation of the young people after laminectomy from the point of molecular biology After cervical vertebra Methods: 1.24 3-month-old goats were randomly divided into two groups: the laminectomy group (n = 12), and the control group (n = 12). TUN-mediated d-UTP nick end-end labeling technique (TUN) mediated by transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase (TdT) EL) to verify the cell apoptosis of the cartilage endplates.2. The separation and culture of primary chondrocytes from 100-150 g SD rats with body weight of 100-150 g SD rats were carried out. Cartilage cells were identified by collagen immunofluorescence staining and the like.0, 0.1, 0.2, 0.5 MPa continuous static pressure were applied to the second generation of chondrocytes, and the morphological changes of the chondrocytes were observed by an inverted phase contrast microscope. The survival rate of the chondrocytes was detected by the CCK-8 method. The flow cytometry And the apoptosis rate of the cartilage cells was detected by the TUNEL method. PK浠，
本文编号：2499516
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