發(fā)布時間：2019-06-14 16:14

【摘要】：研究背景 頸椎板切除術已被廣泛應用于治療兒童脊髓損傷、脊髓腫瘤、先天性畸形以及脊髓空洞癥等疾病的治療。然而,椎板切除手術后可能會發(fā)生脊柱的后凸畸形并最終導致神經(jīng)功能障礙,嚴重影響人類身體健康。因此,深入探討椎板切術后頸椎后凸畸形的發(fā)病機制有著重要科學意義。 在正常情況下,脊柱矢狀面的軸向垂線應當通過頸1(C1)和胸1(T1)中心,頸椎前凸使矢狀面的軸向垂線在C2-C7的后方。頸椎生理前凸的維持主要依靠頸椎前方、后方骨和軟組織結構的完整及力量的均衡。頸椎前方的骨性結構和椎間盤等組織主要承受壓力,后方的關節(jié)突關節(jié)、韌帶和肌肉等組織則主要抵抗頸部張力。先前研究表明：椎板切除術后頸椎后凸畸形的發(fā)生是由于頸椎后伸力矩的破壞所造成的。頸椎板切除術后頸椎后方的肌肉韌帶結構復合體以及骨性結構的完整性遭到了破壞,使得術前后方韌帶所承載的牽張力負荷(后伸力矩)丟失,從而引起前方椎體所承載的壓力性負荷增加并最終導致畸的形發(fā)生。Pal和Sherk等人對頸椎生物力學研究發(fā)現(xiàn)64%的軸向性負荷經(jīng)由兩側的關節(jié)突關節(jié)均勻承載,頸椎椎板切除術后這個軸向性負荷將向前轉(zhuǎn)移,估計將增加前方椎體10倍于原來的壓力性負荷。因青少年椎體生長板正處在生長發(fā)育期,椎板切除術后異常壓力的增加使得生長板軟骨細胞發(fā)育遲滯、凋亡加速,出現(xiàn)椎體楔形變從而形成后凸畸形。 軟骨終板在維系脊柱的生物力學結構完整性以及椎間盤營養(yǎng)方面起重要作用。近年來,異常應力下生長板軟骨細胞凋亡與頸椎后凸畸形的關系逐漸受到重視,對于生長板軟骨細胞凋亡是否參與了椎板切除術后頸椎后凸畸形的發(fā)生過程目前尚未有研究報道。此外,異常應力通過何種信號通路誘導軟骨細胞凋亡亦未被完全闡明。對于這些問題的回答,需要我們做出進一步的深入研究。 目的： 1.建立椎板切除術后青少年頸椎后凸畸形的動物模型,為研究后凸畸形提供有效的研究載體。 2.通過對體外培養(yǎng)的生長板軟骨細胞凋亡機制的研究,以期從分子生物學角度解釋椎板切除術后青少年頸椎后凸畸形的發(fā)病機制。 方法： 1.24只3月齡大山羊隨機分成兩組：椎板切除術組(n=12),和對照組(n=12)。分別于手術前、手術后當天以及手術后3、6個月在麻醉下拍攝標準頸椎側位片,并采用后切線夾角法來評估頸椎曲度的變化。通過透射電鏡法以及末端脫氧核苷酸轉(zhuǎn)移酶(TdT)介導的d-UTP缺口末端標記技術(TUNEL)來驗證軟骨終板的細胞凋亡。 2.取體重100-150g SD大鼠的頸椎體生長板軟骨進行原代軟骨細胞的分離及培養(yǎng),通過軟骨細胞形態(tài)以及二型膠原免疫熒光染色等方法鑒定軟骨細胞。分別給予第二代軟骨細胞施加0、0.1、0.2、0.5MPa持續(xù)靜態(tài)壓力,倒置相差顯微鏡觀察軟骨細胞形態(tài)變化,CCK-8法檢測軟骨細胞存活率,流式細胞儀以及TUNEL法檢測軟骨細胞凋亡率,Western blot法檢測MAPK以及線粒體凋亡相關蛋白表達情況。 結果： 1.體內(nèi)實驗部分： (1)影像學結果： 術前兩組山羊頸椎曲度之間無明顯統(tǒng)計學差異,通過術后與術前X光片比較發(fā)現(xiàn)三個月后椎板切除手術組山羊開始發(fā)生后凸畸形,在第六個月時后凸畸形進一步加重,而對照組山羊頸椎曲度在整個實驗時間內(nèi)并未發(fā)生明顯變化。術前手術組山羊C2-C5平均后切線夾角為-15.8±0.8。,術后三個月降低為19.1±1.1。而到第六個月的時候則為20.2±0.6。。手術后與手術前后切線夾角的變化揭示了后凸畸形的形成。 (2)軟骨細胞凋亡： 在整個觀察時間點內(nèi)對照組生長板僅有少量的凋亡細胞。然而,椎板切除手術組在手術后第三個月即可以觀察到大量凋亡的軟骨細胞(29.7±3.4%),并且凋亡軟骨細胞比例在第六個月時進一步增加(35.6±5.4%),手術組軟骨細胞凋亡率與對照組相比差異具有統(tǒng)計學意義。 (3)透射電鏡觀察： 對照組生長板軟骨細胞呈不規(guī)則形或橢圓形,細胞膜完整,細胞器豐富,染色質(zhì)均勻分布。椎板切除術組可見典型的凋亡的軟骨細胞,表現(xiàn)為細胞膜皺縮,細胞核碎裂,染色質(zhì)濃縮以及細胞器的減少。 2.體外實驗部分： 1.持續(xù)靜態(tài)壓力對終板軟骨細胞活力影響： 為研究持續(xù)靜態(tài)壓力對生長板軟骨細胞存活率的影響,我們分別給予軟骨細胞施加不同靜態(tài)壓力(0,0.1,0.2和0.5MPa)加壓1,6,12,24,36,和48小時后采用CCK-8法檢測細胞存活率。結果顯示軟骨細胞存活率呈壓力以及時間依賴性降低,其中在0.5Mpa大氣壓下作用24小時細胞存活率約為50%。 2.持續(xù)靜態(tài)壓力可以誘導生長板軟骨細胞凋亡： 經(jīng)0.5Mpa大氣壓加壓24小時后,加壓組軟骨細胞TUNEL的陽性率明顯高于對照組,差異具有統(tǒng)計學意義(48.77±4.14%vs.4.43±0.85%,P0.001)。此外我們還采用流式細胞儀AV/PI雙染法來驗證軟骨細胞凋亡,軟骨細胞經(jīng)0.5MPa持續(xù)靜態(tài)壓力下作用24小時后,加壓組軟骨細胞凋亡率較正常組相比有顯著性升高(43.79±4.87%vs.5.66±0.97%,P0.001)。 3.持續(xù)靜態(tài)壓力經(jīng)線粒體及Caspase途徑誘導軟骨細胞凋亡： (1)持續(xù)靜態(tài)壓力對軟骨細胞線粒體功能以及線粒體凋亡相關蛋白影響； 為研究線粒體功能障礙在持續(xù)靜態(tài)壓力誘導生長板軟骨細胞凋亡中的作用,我們采用JC-1免疫熒光染色法來檢測線粒體膜電位(mitochondrial membrane potential,△Ψm)的改變。經(jīng)0.5Mpa大氣壓加壓24小時后,加壓組軟骨細胞紅色熒光強度明顯弱于未加壓組,而綠色熒光強度則明顯高于對照組。這些結果提示加壓組軟骨細胞中線粒體膜電位的降低。此外,實驗組軟骨細胞經(jīng)0.5MPa加壓24小時后Bax蛋白表達水平較對照組明顯升高,而Bcl-2蛋白表達水平則較正常組有顯著性的降低。我們進一步檢測了從線粒體向細胞漿中釋放的細胞色素C的含量,結果顯示加壓后胞漿中細胞色素C的含量明顯增加。 (2)持續(xù)靜態(tài)壓力對軟骨細胞Caspase蛋白表達影響. 我們采用Western blot方法研究Caspase相關蛋白是否參與了持續(xù)靜態(tài)壓力誘導軟骨細胞凋亡過程。結果顯示,在0.5MPa下加壓24小時后實驗組較對照組相比活化的Caspase-9以及Caspase-3蛋白均明顯升高,差異具有統(tǒng)計學意義。應用Caspase-3抑制劑后活化形式的Caspase-3蛋白表達量明顯降低,而且可以顯著性的降低持續(xù)靜態(tài)壓力誘導的軟骨細胞凋亡。 4. MAPK信號通路在持續(xù)靜態(tài)壓力誘導的生長板軟骨細胞凋亡中的作用： 0.5Mpa持續(xù)靜態(tài)壓力刺激生長板軟骨細胞24小時后,磷酸化JNK, p38MAPK和ERK1/2蛋白表達水平較未加壓組有顯著性的升高。與單純加壓組相比,使用10μM SP600125,10μM SB203580和20μM PD98059預處理軟骨細胞后再給予持續(xù)靜態(tài)壓力可以顯著性的降低加壓所引起的磷酸化JNK, p38MAPK和ERK1/2蛋白水平的表達。為了進一步研究MAPK在持續(xù)靜態(tài)壓力誘導軟骨細胞凋亡中的作用,我們加入相應的MAPK抑制劑提1小時處理軟骨細胞后再給予持續(xù)加壓。結果顯示應用10μM SP600125,10μM SB203580和20μM PD98059能夠顯著性的降低持續(xù)靜態(tài)壓力誘導的軟骨細胞凋亡。此外,提前1小時使用10μM SP600125,10μM SB203580和20μMPD98059預處理軟骨細胞可以改善持續(xù)靜態(tài)壓力誘導的線粒體膜電位的降低。Western blot結果顯示提前1小時使用MAPK相應抑制劑預處理軟骨細胞可以上調(diào)Bcl-2以及下調(diào)Bax蛋白表達水平,并減少細胞色素C從線粒體中的釋放。這些結果顯示持續(xù)靜態(tài)壓力經(jīng)MAPK-線粒體信號通路誘導終板軟骨細胞凋亡。 結論： 1.椎板切除術組生長板軟骨細胞凋亡率明顯高于對照組,提示生長板軟骨細胞凋亡可能在椎板切除術后頸椎后凸畸形的形成過程中起重要作用。 2.持續(xù)靜態(tài)壓力可以誘導大鼠頸椎體生長板軟骨細胞凋亡。 3.持續(xù)靜態(tài)壓力經(jīng)由線粒體和Caspase途徑誘導軟骨細胞凋亡。 4. MAPK信號通路參與了持續(xù)靜態(tài)壓力誘導的終板軟骨細胞凋亡過程。
[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，

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