【作者】 许鹏；

【导师】 叶晓健；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 目的后纵韧带骨化症(OPLL)是后纵韧带异位骨化形成,压迫脊髓或神经根后出现脊髓损害或神经根刺激的症状,多发于颈椎,胸椎较少,鲜见于腰椎。其发病机制尚未完全清楚。临床研究发现,颈椎后路减压术后,OPLL患者后纵韧带骨化的发展进程加快,可能与术后颈椎稳定性被破坏有关。同时,国内外研究证实,在应力刺激下后纵韧带成纤维细胞具有成骨能力,碱性磷酸酶(ALP)、I型胶原(COL I)和骨钙素(OC)等表达升高,其机制仍在研究中。因此,探讨应力刺激对OPLL韧带细胞的影响和分子机制,对于我们了解该疾病的发生、发展机制有重要意义。ERK 1/2是MAPK家族的重要成员,阻断其上游MEK后将影响OPLL韧带细胞的成骨分化,同时ERK 1/2磷酸化水平也将发生变化。因此,ERK 1/2磷酸化水平可能在颈椎后纵韧带骨化形成及进展过程中发挥着重要的作用。磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路可以促进细胞增殖、分化以及抑制细胞凋亡,其可能在糖尿病患者OPLL发生过程中起重要作用,而且,机械应力刺激可以激活此通路,那么,AKT蛋白可能也在OPLL形成过程中发挥着重要作用。方法选取2009年8月至2010年8月期间骨科住院病人,16例颈椎后纵韧带骨化和16例颈椎外伤的患者分别行颈前路手术治疗,术中切取韧带标本,无菌条件下置入装有生理盐水的无菌试管内,冰块保护下迅速送往细胞培养室。所有标本均采用自行改良的“组织块贴壁法”培养,将第3代细胞行细胞HE染色和波形蛋白鉴定。分别将两组(OPLL组与NOPLL组)第5代细胞接种于BioFlex 6孔板上,1%胎牛血清(FBS)同步化24h.采用美国FlexerCell公司生产的Flexercell4000细胞加载培养系统进行应力加载,设置参数为牵拉频率0.5 Hz,幅度10%,应力加载时间分别为6h和12h,以静止细胞作为对照,提取细胞总RNA和蛋白,应用real-time PCR和Western-blot技术分别检测两组细胞ALP、COL I、OC及核心转录因子(Runx2)表达量和ERK 1/2及AKT磷酸化水平的差异。然后,分别应用ERK 1/2及AKT蛋白上游阻断剂U0126和LY294002,以证实其作用机制。结果应用自行改良的组织块转瓶贴壁法培养,细胞生长迅速,状态良好。培养7-9天后发现细胞从组织块周围爬出,镜下观察见韧带细胞呈多角形或梭形,细胞沿组织块周围排列。细胞HE染色胞浆粉红色染色较好,细胞核染色较淡;波形蛋白鉴定细胞呈多角形、梭形和圆形,细胞核呈圆形,较大,蓝染,胞浆为绿色,经鉴定为成纤维细胞。培养的细胞纯度较高,没有非成纤维细胞的污染。对两组细胞施加应力刺激后,对ALP、COL I、OC和Runx2的基因表达量进行检测发现,牵拉刺激后OPLL组升高较明显,12h组较静止组明显上调,差异具有统计学意义。而NOPLL组无明显变化。应用阻断剂后,OPLL组三个成骨指标RNA水平较未加阻断剂组下降,差异有统计学意义。结论改良组织块培养法较传统的培养方法有一定优势。后纵韧带细胞呈多角形、梭形,胞浆内波形蛋白阳性表达。机械应力刺激可促进OPLL细胞成骨特异指标的表达,同时激活MEK/ERK1/2和PI3K/AKT信号通路。阻断上述通路后可抑制细胞ALP、COL I、OC的表达。这些结果说明应力刺激通过MEK/ERK1/2和PI3K/AKT调节OPLL细胞的成骨分化,ERK1/2和AKT蛋白的上调在后纵韧带骨化进展过程中的有重要作用。更多还原

【Abstract】 Objective Ossification of the posterior longitudinal ligament (OPLL) is a pathological condition of spinal cord or nerve root compression caused by ectopic bone formation in the spinal ligament, which frequently occurs in cervical spine, and is seldom founded in thoracic spine and lumbar spine. The mechanism of OPLL development remains unclear. Some clinical studies reveal OPLL should develop after posterior cervical surgery in contrast with anterior cervical surgery. In addition, posterior longitudinal ligament fibroblasts should be osteogenic differentiation induced by the mechanical stress, such as gene expression promotion of alkaline phosphatase (ALP), collagen types I (COL I) and osteocalcin (OC). Therefore, it is very important to elucidate the development mechanism of the disease by exploring the role and mechanism of mechanical stress on spinal ligament cells derived from patients with ossification of the posterior longitudinal ligament. ERK 1/2 is one of MAPK family, which phosphorylation and osteogenic differentiation of OPLL cells should change after its upstream protein MEK blocked. Therefore, we postulated ERK1/2 phosphorylation should play an important role in advancing the progression of OPLL. It is the phosphatidylinositol-3-kinase/ protein kinase B (PI3K/AKT) signaling pathway, which could promote cell proliferation, inhibition of apoptosis and cell differentiation. The signaling pathway could be activated by mechanical stress and it also plays an important role in the onset and progression of OPLL suffered with diabetes mellitus. Then, it is likely to be significant of AKT protein for OPLL formation.Methods Sixteen inpatients presenting with OPLL and sixteen NOPLL underwent anterior decompression between August 2009 and August 2010. Specimens of the posterior longitudinal ligaments which were extirpated carefully intraoperatively were put into an aseptic tubule filled with cold physiological saline and immediately transferred to the lab. All specimens were cultured with the modified“tissue fragment attachment-block”method. Hematoxylin eosin (HE) staining and immunostaining of vimentin were performed on the third passage cells. Fifth passage fibroblasts from OPLL and NOPLL patients were seeded on a BioFlex 6-well plate and incubated in DMEM supplemented with 1% FBS for 24 h. The Flexercell 4000 Tension Plus system (Flexercell International Corporation) was used to stretch the cells with mechanical stress of 10%, 0.5 Hz and respectively lasted 0h, 6h and 12 h. The expressions of RNA (ALP、COLI、OC and Runx2) and protein (ERK 1/2 and AKT) were detected. Then signaling pathway blocker, U0126 and LY294002, were used to clarify the mechanism.Results All specimens were cultured with the modified“tissue fragment attachment-block”method and the isolated cells could proliferate rapidly and well in vitro. Cells were observed around the tissue fragment after 7-10 days culture and exhibited a fibroblast-like, spindle-shaped or polygon-shaped appearance. Cytoplasm was stained into deep pink and nucleus was stained into light blue in HE staining. Vimentin immunostaining of cells exhibited spindle-shaped or polygon-shaped appearance with large, round and blue nucleus and green cytoplasm. Cells were identified as fibroblasts and non-fibroblasts not found. The expressions of ALP, COL I, OC and Runx2 of OPLL cells were positively regulated after stimulation for 12h compared to the resting cells. Meanwhile, the osteogenic genes expression significantly up-regulated compared to the resting cells. However, there were no significant changes observed in NOPLL cells. The osteogenic genes expressions were significantly down-regulated with the signaling pathway blocker compared with the group without signaling pathway blocker.Conclusions The modified“tissue fragment attachment-block”method had some advantages than the traditional one. Posterior longitudinal ligament cells exhibited spindle-shaped or polygon-shaped appearance and Vimentin immunostaining were positive. Mechanical stress could significantly promote the osteogenic genes expression and activate the MEK/ERK1/2 and PI3K/AKT pathway. Meanwhile, the osteogenic genes expressions of ALP、COL I、OC were significantly down-regulated with the signaling pathway blocker. Therefore, mechanical stress could induce osteogenic differentiation of spinal ligament cells derived from OPLL patients via these two signaling pathway, and ERK1/2 and AKT protein play important roles.更多还原