氟化物对人体胸椎黄韧带骨化的细胞学行为特性的影响及相关实验研究
|
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。
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 the
meantime, 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 observered
In 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.
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。
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 the
meantime, 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 observered
In 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.
引文
1. Polgar F.Vberinterakuelle wirbelverkalkung. Fortschr GebRontgenstr Nuklearmed Erganzungs band 1920; 40:292-298.
2. Koizurni M. Three cases of paraplegia proved by ossification of theyellow ligament of the spine. J Jpn Orthop Assoc 1962; 36:335-337.
3. Okada K, Oka S, Tohge K, et al. Thoracic myelopathy caused by ossification of the ligamentum flavum. Spine 1991; 16(3):278-287.
4．王哲，王全平，李新奎．胸椎黄韧带骨化症的诊断与治疗．中国矫形外科杂志 1998；5(2)：104-106．
5．王全平，陆裕朴，李稔生．胸椎管狭窄症22例的病理及手术治疗经验．中华骨科杂志 1993；13(1)：15-18．
6．倪斌，贾连顺，戴力杨等．胸椎黄韧带骨化所致椎管狭窄症的诊断及手术治疗．中国脊柱脊髓杂志 1994；4(2)：56-59．
7. Naffziger HC, Inman V, Saunders JB, et al. Lesions of the intervertebral disc and ligamenta flava. Surg Gynee Obstet 1938;66:288-299.
8. Ramsey RH. The anatomy of the ligamenta flava. Clin orthop 1966;44:129-140.
9. Yong-Hing K, Reilly J, Kirkaldy WH, et al. The ligamentumn flavum. Spine 1976;1:226-234.
10. Yahia HL, Grazon S, Strykowshi H, et al. Ultrastructure of the human interspinous ligament and ligamentumn flavum. Spine 1990; 15(4):262-268.
11. Chazal J, Tanguy A, Bouges M, et al. Biomechanical properties ofspinal ligaments and histological study of the supraspinal ligament intraction. J Biomech 1985; 18(3):167-76.
12. Fracassini AS, Field JM, Smith JW, et al. The ultrastructure and mechanics of elastic ligaments. Jounal of Ultrastructure Research 1977;58:244-251.
13．董忻，潘志轩．棘间韧带腰段的形态特点及年龄变化．中华临床解剖学杂志
1994; 12(1) :3-6．
14． 戴力杨,杜晓冰.黄韧带的组织学观察及其临床意义.中华外科杂志1994;32(7) :410-411
15 Jia LS,Shen Q,Chen DY,et al.Dynamic changes of the cervical ligamenta flavua in hyperextension-hyperflexion movement and their measurements.Chinese Medical Journal 1990; 103(1) :66-70．
16． 戴力杨,黄韧带骨化症,中华外科杂志,1989; 27(2) :99-101．
17． Ohtsuka K,Temyama K,Yanagihara M,et al.An epidemiological survey on ossification of ligaments in the cervical and thoracic spinein individuals over 50 years old.N-S-G-Z 1986;60(11) :1087-1098．
18． Kobayashi S.Ossification of the cervical ligamentum flavum.Surg Neurol 1991;35(3) :234-238．
19． Yonenobu K,Ebara S,FujiwaraK,et al.Thoracic myelopathy secondary to ossification of the spinal ligament.J Neurosurg 1987; 66:511-518．
20． Sakamoto R,Ikata T,Murase M,et al.Comparatives study between Magnetic Resonance Imagingand histopathologic findings inossification or calcification of ligaments.Spine 1991; 16(11) : 1253-1251．
21． Hanakita J,Suwa H,Ohta F,et al.Neuroradilogical examination ofthoracic radiculomyelopathy due to ossification of the ligamentum flavum.Neuroradiology 1990; 32(1) :38-42．
22． Tomita K,Kawahara N,Baba H,et al.Circumspinal decompression for thoracic myelopathy due to combined ossification of posterior longitudinal ligament and ligamenrum flavum.Spine 1990; 15(11) :1114-1120．
23． Yonenobu K,Korkusuz F,Hosono N,etal.Lateral rechotomy for thoracic spinal lesions.Spine 1990; 15(11) :1121-1125．
24． Shiokawa K,Hanakita J,Suwa H,Saiki M.Clinical analysis and prognostic study of ossified ligamentum flavum of the thracic spine.J Neurosurg
2001;94:221-226
25． 呼义文,王全平,王哲,尸体胸椎黄韧带骨的病理学观察,临床与实验病理学杂志; 14(2) : 117-119
26． Williams DM,Gabrielsen TO,Latack JT,et al.Ossfication in the caudal attachments of the ligamentum flavum.Radiology 1982; 145:693-697．
27． Williams DM,Gabrielsen TO,Latck JT,et al.Ossification in the cephelic attachments of the ligamentum flavum.Radiology 1984; 150:423-426．
28． Sato M.Pathological studies on the lumber facet joints.NipponSeikeigeka Gakkai Zashi 1991; 66:1078-1090．
29． Ono K,Yonenobu K,Miyamoto S,Okada K.Pathology of ossification of the posterior longitudinal ligament and ligamentum flavum.Clin-Orthop 1999,359(2) : 18-26．
30 Yonenobu K,Sakou T,Ono K.Ossification of the posterior longitudinal ligament.Springer-Verlag Tokyo 1997,pp54,pp69．
31． 王哲,王全平,李新奎,胸椎黄韧带骨化的超微结构观察,中国脊柱脊髓杂志1998;8(3) :170-171
32． Postachini F,Gumina S,Cinetci G,et al.Ligamenta flava in lumbar disc herniation and spinal stenosis.Spine 1994; 19(8) :917-922．
33． Otoni K,Aihara T,Tanaka A,et al.Ossification of the ligamentum flavum of the thoracic spine in adult kyphosis.Intern Orthop 1986; 10:135-139．
34． Moiel RH,Ehni G,Anderson US,et al.Nodule of the ligamentum flavum as a cause of nerve root compression.J Neurosurg 1967; 27;456-458．
35． Fukuyama S,Nakamura T,Ikeda T,Takagi K.The effect of mechanical stress on hypertrophy of the lumbar ligamentum flavum.J Spinal Disord 1995;8(2) :126-130
36． Kondo S,Onari K,Watanabe K,Hasegawa T.Hypertrophy of the posterior longitudinal ligament is a prodromal condition to ossificatioon.Spine
2001,26(1) : 110-114
37． Maigne JY,Ayral X,Guerin SH,et al.Frequency and size of ossifications in the cadual attachments of the ligamentum flavum of the thoracic spine.Surg Radiol Anat 1992; 14(2) : 119-124
38． Yamauchi T,Taketomi E,Matsunaga S,Sakou T.Bone mineral density in patients with ossification of the posteriior longitudinal ligament in the cervical spine.J Bone Miner Metab 1999,17(4) :296-300
39． Mamada T,Nakmura K,Hoshino Y,Saita K.Bone mineral density in patients with ossification of the posteriior longitudinal ligament.Minimal decrease of bone mineral density with aging.Spine 1997,22(20) :2388-2392
40． 王少波,蔡钦林,党耕町等.弥漫性特发性骨肥厚症(DISH)的手术治疗,中国脊柱脊髓杂志1994;4(2) :52-55．
41． Kowahara E,oda Y,Katsudas S,et al.Microfilamentous type IIcollagen in the hyalinized stroma of the hypertrophied ligamentum flavum.Vichows Arch A pcthol Anat Histopathol 1991; 419:37-42．
42． 王哲,王全平,李新奎,胸椎黄韧带骨化与腰椎黄韧带退变的病理及基本代谢元素的比较研究.中华骨科杂志1998． 18(11) :656-658
43． Tsuyama N.Ossification of the posterior longitadinal ligament of the spine.Clinc Orthop 1984; 184:71-83．
44． Utsinger PD.Diffuse idiopathic skeletal hyperostosis.Clin in Rheu Dis 1985; 11:325-329．
45． Ikegawa S,Kurokawa T,Hizuka N et al.Increase of serum growth hormone-binding protein in patients with ossification of the posterior longitudinal ligament of the spine.Spine 1993; 18(13) :1757-1760．
46． Norisu M.Influense of foods on the posterior longitudinal ligamentof the cervical spine and serum sex hormones.Nippon Seikeigeira GakkaiZasshi 1994; 68(12) :1056-1067．
47 朱宪，朱德民，郭世绂等．代谢性骨病学．天津科学技术出版社 第1版．1989；P24-25，P48-49，P104-105．
48. Zimmerman B. The cellular degeneration due to calcium rentation. Calcified Tissue 1993; S53:52.
49. Chintapalli K. Progressive myelopathy in a 32-year-old man. JAMA 1985; 253(15):2255-2257.
50. Matsui H, Katoh Y, Tsuji H, et al. Untreated hypophosphatemic D-resistant rickets with symotamic Ossification of the yellow ligament. JSpinal Disord 1991;4(1): 110-113.
51. Scichi A, Hoshino Y,OhnishiI, etal. The role of calcium metablisom abnormalities in the development of ossification of the longitudinal ligament of the cervical spine. Spine 1992; 17(3S):S30-S32.
52．李斯钝，吕社民，张矢运．锌与骨代谢．国外医学地理分册 1995；16(3)：100-103．
53．廖自甚．微量元素的环境化学及生物效应．中国环境科学出版社．第1版 1992；P234-236．
54. Muller W, Firsching R. Demonstation of elastic fibers with reagentsfor detection of magnesium. J Anat 1991; 175:195-202.
55. Muller W, Firsching R, Iffland R, et al. Age-related calcium/magnesium in the ligamenta flava. Z-Gerontol 1994; 27(5):328-329.
2. Koizurni M. Three cases of paraplegia proved by ossification of theyellow ligament of the spine. J Jpn Orthop Assoc 1962; 36:335-337.
3. Okada K, Oka S, Tohge K, et al. Thoracic myelopathy caused by ossification of the ligamentum flavum. Spine 1991; 16(3):278-287.
4．王哲，王全平，李新奎．胸椎黄韧带骨化症的诊断与治疗．中国矫形外科杂志 1998；5(2)：104-106．
5．王全平，陆裕朴，李稔生．胸椎管狭窄症22例的病理及手术治疗经验．中华骨科杂志 1993；13(1)：15-18．
6．倪斌，贾连顺，戴力杨等．胸椎黄韧带骨化所致椎管狭窄症的诊断及手术治疗．中国脊柱脊髓杂志 1994；4(2)：56-59．
7. Naffziger HC, Inman V, Saunders JB, et al. Lesions of the intervertebral disc and ligamenta flava. Surg Gynee Obstet 1938;66:288-299.
8. Ramsey RH. The anatomy of the ligamenta flava. Clin orthop 1966;44:129-140.
9. Yong-Hing K, Reilly J, Kirkaldy WH, et al. The ligamentumn flavum. Spine 1976;1:226-234.
10. Yahia HL, Grazon S, Strykowshi H, et al. Ultrastructure of the human interspinous ligament and ligamentumn flavum. Spine 1990; 15(4):262-268.
11. Chazal J, Tanguy A, Bouges M, et al. Biomechanical properties ofspinal ligaments and histological study of the supraspinal ligament intraction. J Biomech 1985; 18(3):167-76.
12. Fracassini AS, Field JM, Smith JW, et al. The ultrastructure and mechanics of elastic ligaments. Jounal of Ultrastructure Research 1977;58:244-251.
13．董忻，潘志轩．棘间韧带腰段的形态特点及年龄变化．中华临床解剖学杂志
1994; 12(1) :3-6．
14． 戴力杨,杜晓冰.黄韧带的组织学观察及其临床意义.中华外科杂志1994;32(7) :410-411
15 Jia LS,Shen Q,Chen DY,et al.Dynamic changes of the cervical ligamenta flavua in hyperextension-hyperflexion movement and their measurements.Chinese Medical Journal 1990; 103(1) :66-70．
16． 戴力杨,黄韧带骨化症,中华外科杂志,1989; 27(2) :99-101．
17． Ohtsuka K,Temyama K,Yanagihara M,et al.An epidemiological survey on ossification of ligaments in the cervical and thoracic spinein individuals over 50 years old.N-S-G-Z 1986;60(11) :1087-1098．
18． Kobayashi S.Ossification of the cervical ligamentum flavum.Surg Neurol 1991;35(3) :234-238．
19． Yonenobu K,Ebara S,FujiwaraK,et al.Thoracic myelopathy secondary to ossification of the spinal ligament.J Neurosurg 1987; 66:511-518．
20． Sakamoto R,Ikata T,Murase M,et al.Comparatives study between Magnetic Resonance Imagingand histopathologic findings inossification or calcification of ligaments.Spine 1991; 16(11) : 1253-1251．
21． Hanakita J,Suwa H,Ohta F,et al.Neuroradilogical examination ofthoracic radiculomyelopathy due to ossification of the ligamentum flavum.Neuroradiology 1990; 32(1) :38-42．
22． Tomita K,Kawahara N,Baba H,et al.Circumspinal decompression for thoracic myelopathy due to combined ossification of posterior longitudinal ligament and ligamenrum flavum.Spine 1990; 15(11) :1114-1120．
23． Yonenobu K,Korkusuz F,Hosono N,etal.Lateral rechotomy for thoracic spinal lesions.Spine 1990; 15(11) :1121-1125．
24． Shiokawa K,Hanakita J,Suwa H,Saiki M.Clinical analysis and prognostic study of ossified ligamentum flavum of the thracic spine.J Neurosurg
2001;94:221-226
25． 呼义文,王全平,王哲,尸体胸椎黄韧带骨的病理学观察,临床与实验病理学杂志; 14(2) : 117-119
26． Williams DM,Gabrielsen TO,Latack JT,et al.Ossfication in the caudal attachments of the ligamentum flavum.Radiology 1982; 145:693-697．
27． Williams DM,Gabrielsen TO,Latck JT,et al.Ossification in the cephelic attachments of the ligamentum flavum.Radiology 1984; 150:423-426．
28． Sato M.Pathological studies on the lumber facet joints.NipponSeikeigeka Gakkai Zashi 1991; 66:1078-1090．
29． Ono K,Yonenobu K,Miyamoto S,Okada K.Pathology of ossification of the posterior longitudinal ligament and ligamentum flavum.Clin-Orthop 1999,359(2) : 18-26．
30 Yonenobu K,Sakou T,Ono K.Ossification of the posterior longitudinal ligament.Springer-Verlag Tokyo 1997,pp54,pp69．
31． 王哲,王全平,李新奎,胸椎黄韧带骨化的超微结构观察,中国脊柱脊髓杂志1998;8(3) :170-171
32． Postachini F,Gumina S,Cinetci G,et al.Ligamenta flava in lumbar disc herniation and spinal stenosis.Spine 1994; 19(8) :917-922．
33． Otoni K,Aihara T,Tanaka A,et al.Ossification of the ligamentum flavum of the thoracic spine in adult kyphosis.Intern Orthop 1986; 10:135-139．
34． Moiel RH,Ehni G,Anderson US,et al.Nodule of the ligamentum flavum as a cause of nerve root compression.J Neurosurg 1967; 27;456-458．
35． Fukuyama S,Nakamura T,Ikeda T,Takagi K.The effect of mechanical stress on hypertrophy of the lumbar ligamentum flavum.J Spinal Disord 1995;8(2) :126-130
36． Kondo S,Onari K,Watanabe K,Hasegawa T.Hypertrophy of the posterior longitudinal ligament is a prodromal condition to ossificatioon.Spine
2001,26(1) : 110-114
37． Maigne JY,Ayral X,Guerin SH,et al.Frequency and size of ossifications in the cadual attachments of the ligamentum flavum of the thoracic spine.Surg Radiol Anat 1992; 14(2) : 119-124
38． Yamauchi T,Taketomi E,Matsunaga S,Sakou T.Bone mineral density in patients with ossification of the posteriior longitudinal ligament in the cervical spine.J Bone Miner Metab 1999,17(4) :296-300
39． Mamada T,Nakmura K,Hoshino Y,Saita K.Bone mineral density in patients with ossification of the posteriior longitudinal ligament.Minimal decrease of bone mineral density with aging.Spine 1997,22(20) :2388-2392
40． 王少波,蔡钦林,党耕町等.弥漫性特发性骨肥厚症(DISH)的手术治疗,中国脊柱脊髓杂志1994;4(2) :52-55．
41． Kowahara E,oda Y,Katsudas S,et al.Microfilamentous type IIcollagen in the hyalinized stroma of the hypertrophied ligamentum flavum.Vichows Arch A pcthol Anat Histopathol 1991; 419:37-42．
42． 王哲,王全平,李新奎,胸椎黄韧带骨化与腰椎黄韧带退变的病理及基本代谢元素的比较研究.中华骨科杂志1998． 18(11) :656-658
43． Tsuyama N.Ossification of the posterior longitadinal ligament of the spine.Clinc Orthop 1984; 184:71-83．
44． Utsinger PD.Diffuse idiopathic skeletal hyperostosis.Clin in Rheu Dis 1985; 11:325-329．
45． Ikegawa S,Kurokawa T,Hizuka N et al.Increase of serum growth hormone-binding protein in patients with ossification of the posterior longitudinal ligament of the spine.Spine 1993; 18(13) :1757-1760．
46． Norisu M.Influense of foods on the posterior longitudinal ligamentof the cervical spine and serum sex hormones.Nippon Seikeigeira GakkaiZasshi 1994; 68(12) :1056-1067．
47 朱宪，朱德民，郭世绂等．代谢性骨病学．天津科学技术出版社 第1版．1989；P24-25，P48-49，P104-105．
48. Zimmerman B. The cellular degeneration due to calcium rentation. Calcified Tissue 1993; S53:52.
49. Chintapalli K. Progressive myelopathy in a 32-year-old man. JAMA 1985; 253(15):2255-2257.
50. Matsui H, Katoh Y, Tsuji H, et al. Untreated hypophosphatemic D-resistant rickets with symotamic Ossification of the yellow ligament. JSpinal Disord 1991;4(1): 110-113.
51. Scichi A, Hoshino Y,OhnishiI, etal. The role of calcium metablisom abnormalities in the development of ossification of the longitudinal ligament of the cervical spine. Spine 1992; 17(3S):S30-S32.
52．李斯钝，吕社民，张矢运．锌与骨代谢．国外医学地理分册 1995；16(3)：100-103．
53．廖自甚．微量元素的环境化学及生物效应．中国环境科学出版社．第1版 1992；P234-236．
54. Muller W, Firsching R. Demonstation of elastic fibers with reagentsfor detection of magnesium. J Anat 1991; 175:195-202.
55. Muller W, Firsching R, Iffland R, et al. Age-related calcium/magnesium in the ligamenta flava. Z-Gerontol 1994; 27(5):328-329.