【作者】 杨海松；

【导师】 陈德玉； 卢旭华； 杨立利；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 目的颈椎后纵韧带骨化(ossification of the posterior longitudinal ligament, OPLL)是一种结缔组织的后纵韧带演变为骨组织的病理状态,这种异位骨形成可持续进展而压迫脊髓和神经根导致患者出现四肢麻木无力等临床症状,严重影响患者生活质量,多见于日本等亚洲国家,发病机制尚不完全清楚。有学者认为后纵韧带成纤维细胞具有成骨细胞活性,比如细胞外矿化结节的形成和骨钙素、碱性磷酸酶活性的增强,其在某种机制的作用下向成骨细胞转化,最终进展成为骨化组织而压迫脊髓和神经根。既往研究亦显示后纵韧带骨化是一种多病因的疾病,有着复杂的基因和环境因素共同作用。临床许多学者发现颈椎后路椎板切除或椎管成形术后由于破坏了颈椎后方结构产生颈椎不稳而导致骨化明显进展,而在前路融合术后,去除了颈椎节段不稳因素,骨化进展明显减慢或完全消失,提示机械应力刺激可能在后纵韧带骨化的发生发展过程中发挥着重要的促进作用。但是后纵韧带骨化进展过程中涉及的信号传导通路目前仍在研究中。缝隙连接是一种连接相邻细胞的跨膜蛋白,允许分子量小于1KD的分子穿透细胞膜从一个细胞进入另一个细胞从而起到信息传递的作用。组成缝隙连接蛋白的亚单位为connexin,目前已经发现20余种connexin缝隙连接蛋白,但在骨组织中分布最广、作用最多的是connexin43 (Cx43)缝隙连接蛋白。这种缝隙连接在促进骨形成的过程中起着重要的作用,据研究发现,在connexin43缺如的成骨细胞,不但其矿化能力缺失而且对生物信号的反应能力亦丧失。因此我们认为在颈椎后纵韧带骨化形成及进展过程中connexin43缝隙连接蛋白有可能发挥着重要的信号传递作用。本试验体外培养颈椎后纵韧带骨化和非骨化患者的韧带细胞,探讨细胞的培养方法,验证后纵韧带骨化组韧带成纤维细胞的成骨活性及机械应力刺激在后纵韧带骨化进展过程中的促进作用,同时证明connexin43缝隙连接蛋白的信号传递作用。方法2008年12月至2009年12月24例颈椎后纵韧带骨化和20例非颈椎后纵韧带骨化患者(颈椎外伤14例,颈椎间盘突出4例及颈椎病2例)行颈前路手术治疗,术中切取所有患者的韧带标本,无菌条件下置入已消毒的含无血清DMEM的标本管内,37℃条件下迅速送往细胞培养室。4例颈椎后纵韧带骨化之标本采用酶消化培养法,其余40例标本采用组织块培养法进行细胞体外培养。采用免疫细胞化学及免疫荧光技术检测所培养细胞胞浆内波形蛋白从而进行细胞鉴定。将传至第3代的两组细胞(OPLL组与non-OPLL组)接种于6孔培养板内,贴壁后同步化24h,提取两组细胞的总RNA和蛋白,采用半定量RT-PCR及Western blotting技术检测两组细胞三个成骨特异指标骨钙素(OCN)、碱性磷酸酶(ALP)及Ⅰ型胶原(COL I)和缝隙连接蛋白Cx43表达量的差异。采用美国Flexercell公司生产的Flexercell4000细胞加载培养系统对OPLL组及non-OPLL组第3代细胞进行机械应力刺激,分别在应力刺激前,刺激后12h和24h三个时间点提取细胞总的RNA和蛋白,分别检测两组细胞在三个不同时间点OCN、ALP、COL I及Cx43表达量的差异。设计合成针对Cx43的特异siRNA序列,采用Lipofectamine 2000细胞转染试剂转染于骨化组第三代细胞内,持续转染48h。Western blotting技术检测转染效率,同时采用RT-PCR技术测量转染前后细胞细胞内OCN、ALP、COL I及Cx43之mRNA表达量的差异。然后对转染48h的细胞和对照组细胞进行机械应力刺激,测量两组细胞应力刺激前,刺激后12h和24h细胞表达OCN、ALP、COL I及Cx43表达量的差异变化。结果采用酶消化培养法的4例后纵韧带骨化之标本在酶消化完成后可见游离的圆形细胞,次日于倒置相差显微镜下观察见圆形的细胞变为细胞碎片,细胞死亡,采用酶消化培养法培养细胞失败。对其余40例标本采用组织块培养法进行细胞培养,培养7—10天后发现组织块周围细胞爬出,镜下观察见细胞呈梭形、纺锤形及多角的星形,细胞排列紊乱。伴随细胞的分裂生长,细胞排列逐渐有序化,培养20天左右呈“栅栏状”排列。采用免疫细胞化学及免疫荧光技术检测细胞内波形蛋白发现呈阳性表达(表现为红色和绿色荧光)。采用RT-PCR对OPLL组和non-OPLL组细胞内OCN、ALP、COL I的mRNA表达量进行检测发现,前者较后者明显上调,Cx43缝隙连接蛋白的mRNA和蛋白表达量亦有明显上调,差异具有统计学意义。对OPLL组第3代细胞内Cx43进行siRNA干扰48h,Western blotting检测转染效率达69%,同时检测转染后三个成骨特异指标mRNA的表达量,发现其出现了明显的下调,差异亦有统计学意义。对OPLL组和non-OPLL组细胞进行机械牵张应力刺激24h后发现,OPLL组细胞表达OCN、ALP、COL I及Cx43的量明显上调,而non-OPLL组无明显变化。对骨化组细胞Cx43缝隙连接蛋白转染48h后再次进行机械牵张应力刺激24h,发现细胞OCN、ALP、COL I的mRNA表达量不再上调,差异无统计学意义。结论组织块培养法可成功进行颈椎后纵韧带成纤维细胞的培养,细胞呈梭形、纺锤形及多角的星形,胞浆内波形蛋白阳性表达。OPLL组细胞OCN、ALP及COL I的高表达说明其潜在的成骨活性。机械应力刺激可促进OPLL细胞成骨特异指标的表达,验证了机械应力刺激在后纵韧带骨化进展过程中的促进作用。对细胞Cx43缝隙连接蛋白进行siRNA干扰可抑制细胞OCN、ALP及COLⅠ的表达提示Cx43缝隙连接蛋白在后纵韧带骨化进展过程中的信号传递作用。更多还原

【Abstract】 Objective Ossification of the posterior longitudinal ligament(OPLL) is a pathological condition causing ectopic bone formation in the cervical spinal ligament and is a common disease in Japan and throughout Asia. Although the mechanism of OPLL development remains unclear, genetic factors and local factors have been proposed and partly confirmed. OPLL can compress the spinal cord and its roots, leading to various degrees of neurological symptoms from discomfort to severe myelopathy. However, it is known that the presence of OPLL does not always indicate the presence of cervical myelopathy. OPLL is a chronic and progressive disease, and clinical symptoms may only occur after the ossified ligaments develop to a certain degree. Although some studies have investigated the osteogenesis of fibroblasts from ligaments of OPLL and the role of mechanical stress in OPLL development, the mechanism of cellular signaling transduction remains unclear. Gap junctions are pores or channels that span the cellular membrane and allow molecules less than 1 kD to pass from one cell to another, providing bilateral communication between cellular cytoplasms. These junctions are formed by two hexameric hemichannels, composed of 6 protein subunits, termed connexins. There are more than 20 identified mammalian connexins, and the most abundant connexin family member present in bone cells is connexin43 (Cx43). This mode of cell-cell communication is of particular importance in the skeleton, where a large variety of systemic and locally generated signals are transduced into biological signals and transmitted to cells at specific locations to permit bone formation. Mineralization in Cx43 null osteoblasts was reported to be defective, and osteoblast responses to anabolic signals were flawed. Thus, we hypothesized that the mRNA and protein expression of the Cx43 gap junction in spinal ligament fibroblasts derived from OPLL patients may be up-regulated and play a key role in the signal transmission from one cell to another, thereby promoting the development of OPLL. To explore this possibility, we evaluated the different expressions of osteoblastic genes (osteocalcin (OCN), alkaline phosphatase (ALP) and type I collagen (COL I)) and the Cx43 gap junction via RT-PCR in cells cultured from spinal ligament specimens from OPLL and non-OPLL patients. Mechanical stress was loaded on OPLL ligament fibroblasts, and the above mentioned gene expressions were compared before the application of mechanical stress and again at 12 h and 24 h after stress loading. Additionally, we performed siRNA interfering targeting Cx43 gap junction and again evaluated the above mentioned gene expressions.Methods Twenty-four patients presenting with OPLL and 20 non-OPLL patients underwent anterior decompression between December 2008 and December 2009. Specimens of the posterior longitudinal ligaments were collected intraoperatively. Enzymatic digestion culture was performed on four OPLL ligament specimens, and tissue fragment culture was performed on the remaining 40 specimens. Inverted phase contrast microscopy and hematoxylin eosin (HE) staining were used to observe cell morphology. The mouse anti-vimentin antibody was used to identify the cultured cells via immunocytochemistry and immunofluorescence (ICC/IF). Fibroblasts from OPLL patients were preloaded with mechanical stress using a Flexercell 4000 Tension Plus system. The mRNA expressions of osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (COL I) and Cx43 were detected in OPLL and non-OPLL cultures, at pre-stress and at 12 and 24 h after stress loading by semi-quantitative RT-PCR. The protein expression of the Cx43 gap junction was also detected via Western blotting. Small interfering RNA (siRNA) targeting the Cx43 gap junction was transfected into the fibroblasts derived from the OPLL patients using Lipofectamine 2000 Reagent. The expressions of the indexes mentioned above were compared between the negative control group and the transfection group, and between groups before and after stress loading.Results No adherent cells were found in the four specimens of the enzymatic digestion culture; however, in the remaining 40 specimens that underwent tissue fragment culture, cultivated cells were observed 7-10 days after culture. Inverted phase contrast microscopy revealed a more hypertrophic cell appearance and cell matrix in the OPLL group. However, the cells were more orderly in the non-OPLL group. HE staining showed fusiform and multi-angular star morphologies, large and elliptical cell nuclei and ill-defined cell appearances. ICC/IF exhibited positive results of vimentin staining. The expressions of OCN, ALP, COL I and Cx43 from OPLL ligament fibroblasts were greater than those from non-OPLL cells as measured by RT-PCR and Western blotting. The same results were obtained after 12 h and 24 h of mechanical stress loading as compared with cells without stress. Specific siRNA that targeted the Cx43 gap junction reduced the mRNA expressions of OCN, ALP and COL I remarkably. The expressions of the osteoblastic genes did not up-regulate after 72 h of transfection in OPLL group, even with mechanical stress loading. Conclusions Tissue fragment culture of the cervical posterior longitudinal ligament provided a successful fibroblast culture, showing good adherence and subculture. The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, and applied mechanical stress may aid in the progression of OPLL. In these processes, the Cx43 gap junction played an important role.更多还原