【作者】 王哲；

【导师】 王全平；

【作者基本信息】 第四军医大学 ， 外科学， 2002， 博士

【摘要】 胸椎黄韧带骨化症是一种特殊的异位骨化形式，是目前常见的一种临床疾患，临床上多表现为胸椎管狭窄所引起的脊髓压迫症状，严重者可导致截瘫。有关其发病机制的研究尽管国内、外文献报道较多，目前仍不清楚，因而尚无预防可言。目前在胸椎OLF的诊断中，MRI加CT是公认有效的检查方法，但价格昂贵。胸椎OLF的保守治疗效果差，临床治疗仍以后路减压术为主，研究表明，诊断延误与OLF多发性是造成患者术后功能恢复差的重要原因。因此，如何通过其病因学研究，及早发现黄韧带骨化的高危人群，及时诊断、治疗与预防，是目前黄韧带骨化研究的关键。 在异位骨化中，能够引起细胞增殖、分化与基质合成的骨生长因子起重要作用。为此本课题首先通过免疫组织化学的方法，观察了BMP-2、TGF-β1及Ⅰ型、Ⅱ型胶原蛋白在人胸椎骨化黄韧带中的定位与表达。结果表明，在骨化黄韧带中，BMP-2与TGF-β1在未钙化软骨细胞均可见阳性表达；同时，BMP-2在成纤维细胞样细胞与新生小血管周围的间充质细胞亦可见阳性表达，提示BMP-2在黄韧带骨化中可能通过诱导未分化间充质细胞分化，在骨诱导与早期软骨发生中起始动因素的作用；而TGF-β1则可能在稍后的骨化进程中起作用。另外，在骨化黄韧带中未钙化的软骨细胞胞浆及其胞外基质中，同时观察到Ⅰ型与Ⅱ型胶原蛋白的阳性表达，这同由BMP所诱导的软骨细胞胞外基质中Ⅰ型与Ⅱ型胶原蛋白的表达相仿，进一步提示BMP-2在人黄韧带 第四罕医大学博士学位论文 一 骨化中起重要作用。 以往众多研究表明，氟中毒与脊柱韧带骨化，脊柱退行性变与脊柱韧带骨 化关系密切。由于氟化物对人体韧带骨化影响的在体实验研究存在种种困难， 有关氟化物在体外对人体韧带骨化影响的实验研究就显得十分重要，但目前尚 未见有关研究报道。为此我们首先成功地建立了人黄韧带细胞的体外培养模 型，建立了24株不同来源的黄韧带细胞系，并对其特性进行了初步分析：在 此基础上，研究了NaF对人黄韧带细胞系的sP活性与BGP合成的影响。结 果表明，培养的人黄韧带细胞可在体外增殖与传代，来源于不同病变患者的细 胞系表现出不同的表型特征：骨化患者的细胞系主要表现为典型的成骨细胞表 型；退变患者细胞系主要表现为较幼稚的成骨细胞表型：而正常黄韧带细胞系 则为成纤维细胞表型。在体外，高浓度的NaF可导致人黄韧带细胞系发生中 毒反应；低浓度的NaF可导致退变黄韧带细胞系中，表现出由较幼稚成骨细 胞表型向成骨细胞分化、成熟的趋势，而相同浓度下，NaF对人骨化与正常黄 韧带细胞系无明显效应。该结果提示在体内，氟化物可能是通过刺激退变黄韧 带细胞进一步向成骨细胞分化、成熟，诱导黄韧带在退变基础上进一步骨化。 最后，本课题通过使实验大鼠饮水摄入过量NaF，复制出与临床相近的氟 中毒模型，采用X线检查，骨密度分析，血清学检测及组织病理观察等多种 手段，探讨了NaF在实验大鼠腰椎黄韧带病变中的作用。结果表明，NaF可 导致部分实验大鼠的腰椎黄韧带退变、骨化。 总之，本课题首次通过体外模型，观察了NaF对人黄韧带细胞骨化的影 响，结合动物实验，分析了NaF在致实验大鼠腰椎黄韧带退变、骨化过程中 的作用。结果表明，过量NaF在体内、外实验中均呈现出诱导黄韧带骨化发 生的趋势。这一结果提示需进一步进行相关的临床与实验研究，从而为寻求黄 韧带骨化的早期预防、诊断与治疗措施奠定基础。更多还原

【Abstract】 Osisfication of the ligamentum flavum (OLF) of the thoracic spine is a special kind of ectopic ossification, and can be found frequently in spinal diseases. It may cause compression myelopathy, even paraplysis. The etiology of OLF still remains unclear, MRI and CT are effective examination in OLF, but too expensive. The un-operative treatments for OLF are often uneffetive, and laminectomy still is the main procedure hi treatment. The previous studies show that the main reasons for poor post-operation results of OLF patients are the delation in diagnosis and the needs of continued operation in OLF in spine. So how to find the high risk group of OLF all in good time and its’ preventive treatment is the key to the study of the etiology of OLF.As we know, Bone growth factors play important roles in ectopic ossification. We immunochemically detected the expressions of BMP-2, TGF-pl and type I , II collagen in tissue samples of ligamentum flavum from OLF patients. The results showed that BMP-2 and TGF-pl were present in the uncalcified chondrocytes, and BMP-2 was also present in the fibroblast-like cells near cartilaginous area and in mesenchymal cells around new-born small vessels, which implied that BMP-2 play an important role in early process of OLF and TGF-pl in late process. In themeantime, the cytoplasm and extracellular matrix in the uncalcified chondrocytes were stained intensively for type I, II collagen, also implying that BMP-2 play an important role in OLF .Many reports shows there is a close relationship between fluorosis and ossification of the spinal ligaments, and also between degeneration and ossification of the spinal ligaments. Because of the limitations in study of fluoride effect on ossification of the spinal ligaments in vivo, the study in vitro is desperately needed and important. Such kinds of study has never been reported. Therefore we succesefully estibalished twenty-four cell lines obtained from ligamentum flavum from patients with OLF, lumbar stenosis and control. The characterization of these cultrued cells were analysised. Then we study the effects of fluoride on these cells. The results showed that the cultrued human ligamentum flavum cells could proliferate and be passed in vitro. Cells from OLF patients showed typical phenotype of osteoblast, while cells from patients with lumbar stenosis showed phonetypic characteristics for osteoblast in earlier process, and cells from control subjects with normal ligamentum flavum showed typical fibroblast phonetype. We also found that fluoride at high concentration could lead to cytotoxicity of the cultrued human ligamentum flavum cells in vitro, however, lower concentration resulted in significant increase of ALP activity and elevation of osteocalcin of most of cell lines from patients with lumbar stenosis, while made no effect on most of cell lines from patients with OLF and control subjects. The result suggested fluoride may cause OLF in vivo through stimulations of cells with earlier osteoblast-like phonetype into typical osteoblasts.During the study we also reproduced the model of fluorosis in rats by feeding with water of high concentration fluoride.The effects of fluoride on leading to degeneration and ossification of the ligamentum flavum in rat’s lumbar spine were observeredIn conclusion, for the first time we conducted the experimental study of the effects of fluoride on cultrued human ligamentum flavum cells in vitro, and also degeneration and ossification of the ligamentum flavum in rat’s lumbar spine caused by fluoride in vivo. The present study demonstrated the definite effect of fluoride on the ossification of ligamentum flavum in vivo and vitro, and indicated that continued research in clinics and experiments may be needed, in order to find a simple and effective method in early diagnosis, treatment and prevention of the OLF.更多还原