脊髓损伤后骨质疏松发病机制及Bcl-2对成骨细胞凋亡影响的实验研究
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摘要
研究背景：骨质疏松(Osteoporosis,OP)是脊髓损伤(Spinal cord injury,SCI)后重要的并发症之一，它所引起的损伤节段以下迅速的骨量丢失以及骨组织生物力学强度的下降导致SCI患者病理性骨折发生率较高，文献报道达20％左右，这给患者康复带来诸多不便。目前对于SCI后骨质疏松临床方面的报道较多，但对于动物实验方面的研究甚少。动物模型建立对于病理机制认识和疾病防治有重要价值。
第一部分实验目的：对SCI后大鼠骨转换状态、骨密度变化、骨组织生物力学特点进行分析，评估大鼠作为SCI后骨质疏松动物模型的可行性；对SCI后骨质疏松的病理机制进行相关研究。方法：切断SD大鼠胸髓建立SCI动物模型；在SCI后不同时间段对血尿尘化、骨密度、股骨颈生物力学进行测定；对局部骨组织的超微结构变化进行观察；应用免疫组织化学结合计算机图象分析对局部松质骨内细胞因子TNF-α、BMP、IL-6的表达改变进行分析。结果：SCI大鼠伤后六周膀胱内有大量结石形成。血钙、尿钙、尿钙／肌酐在伤后一、三、六周均升高(P<0.05，0.01，0.05；P<0.001，0.001，0.01；P<0.05，0.01，0.01)；血ALP在伤后一周时下降差异有显著性意义(P<0.01)，三、六周时恢复正常。胫骨近端，股骨远、近端的骨密度(Bone mineral density，BMD)在六周时较对照组下降且差异显著(P<0．05，0．01，0．05)。六周时SCI组股骨近端最大载荷，结构刚度和能量吸收均低于对照组且有显著性差异(P<0．01，0．05，0．05)。超微结构观察发现SCI后一周成骨细胞功能低下表现为内质网及高尔基体稀少，线粒体空泡化；一些正在向骨细胞转化的成骨细胞出现异染色质浓聚呈月环状边集于核膜下等凋亡样改变。SCI后三周成骨细胞与对照组比较无显著差异。伤后三周SCI组骨细胞
— ———————
’有四种改变：一是细胞器减少，骨陷窝增大，絮状物质增多，凝聚以及骨陷窝壁
嗜饿板层形成等骨细胞性溶骨表现；二是退变相骨细胞表现为胞质和细胞器出现
严重囊性改变如线粒体空泡化，细胞壁不完整，细胞器不清晰。三是典型核固缩，
细胞处于崩解状态为坏死的形态学改变。四是异染色质凝集、边聚，细胞出芽起
泡等典型凋亡样改变。细胞因子表达变化的结果表明SCI后一周TNF．口阳性染色
强度显著高于对照组p＜0．0引，六周时与对照组相比无显著性差异。＊＊P表达在
一周时与对照组无显著差异，六周时表达显著增强p＜O．0引，1＊－6的表达在一周
时与对照组无显著性差异，六周时表达强度明显高于对照组，差异显著①＜O．0引。
结论：ISCI后大鼠六周发生骨质疏松，骨代谢特点为高转换型；SCI大鼠在骨转
换状态、骨质疏松部位以及骨折相对危险性增加方面与人类有较好相似性，可以
初步作为SCI后骨质疏松的动物模型。2 SCI后成骨细胞功能低下是早期成骨代
谢低下的重要原因：骨细胞的溶骨、退变、坏死和凋亡样改变可能是骨代谢偶联
失调的细胞学基础。3细胞因子 iFQ早期升高，IL6和 BMP在晚期升高，提
示它们在SCI后骨质疏松发病的不同阶段起作用。TNF－口可能与伤后早期成骨代
谢降低有关：1卜6则是晚期破骨代谢增强的重要因素：8＊P可能是伤后骨转换加
快的病理基础。
研究背景：骨质疏松症发生与骨重建改变有关。骨重建过程中骨形成的多少
山具有成骨活性的成骨细胞的数量决定，成骨细胞过度凋亡致成骨细胞数量减少
是骨质疏松病理过程中成骨代谢降低的重要环节。了解成骨细胞凋亡的基回调控
对未来骨质疏松基因治疗有积极作用。
第二部分实验目的：探讨骨质疏松组织中高表达的TNF Q在体外诱导成骨细
胞凋亡过程中凋亡相关基因BclZ／Bax蛋白表达变化及意义。方法：体外原代培养
大鼠颅骨成骨细胞：加入0，10，20，30，40ng／ml hFQ作用 24小时诱导凋亡；观
察不同浓度h卜。对k卜2、8。蛋白表达的影响：k1．Z、＊。蛋白表达与诱导
f 凋亡的相关性分析。结果：TNFQ剂量依赖性地刺激大鼠成骨细胞凋亡：浓度为
0刁0ng／ml时剂量依赖性地升高Bax的表达，30＊0ng加l时降低Bax表达；浓度在
10ng／ml时刺激 BclZ 的表达，而 20－40ng／ml时降低 BclZ的蛋白表达山ax与 Bcl－2
3
— —————
一
相对表达强度与细胞的死亡呈正相关（n—25，，0．827308，P＜0刀of），结论：
Bcl－2／Bax蛋白相对比例下调是TNF－口诱导成骨细胞发生凋亡过程中的一个重要
病理环节。
研究背景：多项研究表明k－2、Bax蛋白相对含量是不同因素诱导成骨细胞
凋亡过程中一个共同的病理环节，那么是否B。l－2过度表达对成骨细胞生存起保
护作用呢？细胞凋亡发生的核心机制是蛋白溶解系统的活
Background: Osteoporosis is one of complications due to spinal cord injury (SCI). Osteoporosis after SCI lead to increased bone mass loss as well as decreased biomechanics quality which often result in pathologic fractures below the injured level with the incidence of 20% or so reported by some researchers. Nowadays there are so much research works on clinical practice, and little attention focus on experimental research. The establishment of the animal model would contribute to the investigation of pathogenesis. prevention and treatment for this disease.
The aim of the first part of study is to evaluate the possibility of rats with SCI being used as an experimental model of osteoporosis after SCI by examining the bone turnover status, bone mineral density and biomechanical competence of the bone. Also, pathogenesis mechanism of osteoporosis due to SCI was investigated. Method: SCI model was established by cutting off the distal thoracic spinal cord of rats; The investigation of biochemical values relating to bone resorption and formation, bone mineral density and biomechanics were performed; Ultrastructural investigation of bone were performed; BMP, IL-6 and TNF- a proteins in the bone were examined by using immunochemical stain combined with the imaging analysis techniques. Results: Calculus of the bladder was found in the SCI animals 6 week after injuries. Biochemical date demonstrated serum calcium, urine calcium, and urine calcium/creatinine rose dramatically compared with control groups at week 1,3,6.
(PO.05, 0.01, 0.05; PO.001, 0.001, 0.01; PO.05, 0.01, 0.01, respectively). Serum total alkaline phosphates dropped significantly at week 1 (P<0.01), and then returned to normal level at week 3,6. Bone mineral density measured by dual energy x-ray absorption at proximal of tibia, distal and proximal of femur showed a significant fall(P<0.05, 0.01, 0.05). There are significant decrease in value of maximum load, energy absorption capacity and structural rigidity in femoral neck through bending test after 6 weeks (P<0. 01, 0.05, 0.05). Ultrastructural investigation revealed little endoplasmic reticulum and Golgi apparatus were found in osteoblast and vacuole changes of mitochondrion indicating the low activity of osteoblast. Also, condensation of heterochromatin collected under the edge of nuclear membrane indicating the apoptosis were also found in osteoblasts. No differences were found between the SCI and control group in osteoblast ultrastructure at week 3. There were four basic changes in ultrastructure of osteocytes as follows. First, osteocytic dissolve characterized by decreased organelles. enlargement of bone lacuna, increasing concentrated wadding material and forming of osmium -favored stratum. Second, degenerative osteocytes revealed decreased organelles. sake-like change and some cells without integrated wall. Third, some typical necrosis cell showed nuclear pycnosis and cell collapsed. Fourth, changes such as bubble and bud. heterochromatin condensation, edge collection of heterochromatin under the nuclear membrane suggested apoptotic osteocytes. The expression of tumor necrosis factor-a (TNF-a) rose at week 1 significantly and returned to normal at week 6. Immunochemistry stain of bone morphogenetic protein (BMP) and interleukin-6 (IL-6) were more intensive in SCI groups at week 6 than that in control groups (PO.05, 0.05,respectfully), however, no difference was found between two groups at week 1. Conclusions: Osteoporosis develops at week 6 in rats suffering from SCI. Rats underwent SCI have a similar changes in bone turnover, bone loss site and the risk of fracture, so could be used as the animal model for osteoporosis after SCI. Decreased vigor and increased possibility of fall of total live osteoblasts resulted from
the apoptosis may contributed to the decreased bone forming ability in early state. Dissolve, degeneration, necrosis and apoptosis of osteocytes are the probable causes for decoupling. The data collected from the alteration of the immunochemistry indicated that cytokine play
第一部分实验目的：对SCI后大鼠骨转换状态、骨密度变化、骨组织生物力学特点进行分析，评估大鼠作为SCI后骨质疏松动物模型的可行性；对SCI后骨质疏松的病理机制进行相关研究。方法：切断SD大鼠胸髓建立SCI动物模型；在SCI后不同时间段对血尿尘化、骨密度、股骨颈生物力学进行测定；对局部骨组织的超微结构变化进行观察；应用免疫组织化学结合计算机图象分析对局部松质骨内细胞因子TNF-α、BMP、IL-6的表达改变进行分析。结果：SCI大鼠伤后六周膀胱内有大量结石形成。血钙、尿钙、尿钙／肌酐在伤后一、三、六周均升高(P<0.05，0.01，0.05；P<0.001，0.001，0.01；P<0.05，0.01，0.01)；血ALP在伤后一周时下降差异有显著性意义(P<0.01)，三、六周时恢复正常。胫骨近端，股骨远、近端的骨密度(Bone mineral density，BMD)在六周时较对照组下降且差异显著(P<0．05，0．01，0．05)。六周时SCI组股骨近端最大载荷，结构刚度和能量吸收均低于对照组且有显著性差异(P<0．01，0．05，0．05)。超微结构观察发现SCI后一周成骨细胞功能低下表现为内质网及高尔基体稀少，线粒体空泡化；一些正在向骨细胞转化的成骨细胞出现异染色质浓聚呈月环状边集于核膜下等凋亡样改变。SCI后三周成骨细胞与对照组比较无显著差异。伤后三周SCI组骨细胞
— ———————
’有四种改变：一是细胞器减少，骨陷窝增大，絮状物质增多，凝聚以及骨陷窝壁
嗜饿板层形成等骨细胞性溶骨表现；二是退变相骨细胞表现为胞质和细胞器出现
严重囊性改变如线粒体空泡化，细胞壁不完整，细胞器不清晰。三是典型核固缩，
细胞处于崩解状态为坏死的形态学改变。四是异染色质凝集、边聚，细胞出芽起
泡等典型凋亡样改变。细胞因子表达变化的结果表明SCI后一周TNF．口阳性染色
强度显著高于对照组p＜0．0引，六周时与对照组相比无显著性差异。＊＊P表达在
一周时与对照组无显著差异，六周时表达显著增强p＜O．0引，1＊－6的表达在一周
时与对照组无显著性差异，六周时表达强度明显高于对照组，差异显著①＜O．0引。
结论：ISCI后大鼠六周发生骨质疏松，骨代谢特点为高转换型；SCI大鼠在骨转
换状态、骨质疏松部位以及骨折相对危险性增加方面与人类有较好相似性，可以
初步作为SCI后骨质疏松的动物模型。2 SCI后成骨细胞功能低下是早期成骨代
谢低下的重要原因：骨细胞的溶骨、退变、坏死和凋亡样改变可能是骨代谢偶联
失调的细胞学基础。3细胞因子 iFQ早期升高，IL6和 BMP在晚期升高，提
示它们在SCI后骨质疏松发病的不同阶段起作用。TNF－口可能与伤后早期成骨代
谢降低有关：1卜6则是晚期破骨代谢增强的重要因素：8＊P可能是伤后骨转换加
快的病理基础。
研究背景：骨质疏松症发生与骨重建改变有关。骨重建过程中骨形成的多少
山具有成骨活性的成骨细胞的数量决定，成骨细胞过度凋亡致成骨细胞数量减少
是骨质疏松病理过程中成骨代谢降低的重要环节。了解成骨细胞凋亡的基回调控
对未来骨质疏松基因治疗有积极作用。
第二部分实验目的：探讨骨质疏松组织中高表达的TNF Q在体外诱导成骨细
胞凋亡过程中凋亡相关基因BclZ／Bax蛋白表达变化及意义。方法：体外原代培养
大鼠颅骨成骨细胞：加入0，10，20，30，40ng／ml hFQ作用 24小时诱导凋亡；观
察不同浓度h卜。对k卜2、8。蛋白表达的影响：k1．Z、＊。蛋白表达与诱导
f 凋亡的相关性分析。结果：TNFQ剂量依赖性地刺激大鼠成骨细胞凋亡：浓度为
0刁0ng／ml时剂量依赖性地升高Bax的表达，30＊0ng加l时降低Bax表达；浓度在
10ng／ml时刺激 BclZ 的表达，而 20－40ng／ml时降低 BclZ的蛋白表达山ax与 Bcl－2
3
— —————
一
相对表达强度与细胞的死亡呈正相关（n—25，，0．827308，P＜0刀of），结论：
Bcl－2／Bax蛋白相对比例下调是TNF－口诱导成骨细胞发生凋亡过程中的一个重要
病理环节。
研究背景：多项研究表明k－2、Bax蛋白相对含量是不同因素诱导成骨细胞
凋亡过程中一个共同的病理环节，那么是否B。l－2过度表达对成骨细胞生存起保
护作用呢？细胞凋亡发生的核心机制是蛋白溶解系统的活
Background: Osteoporosis is one of complications due to spinal cord injury (SCI). Osteoporosis after SCI lead to increased bone mass loss as well as decreased biomechanics quality which often result in pathologic fractures below the injured level with the incidence of 20% or so reported by some researchers. Nowadays there are so much research works on clinical practice, and little attention focus on experimental research. The establishment of the animal model would contribute to the investigation of pathogenesis. prevention and treatment for this disease.
The aim of the first part of study is to evaluate the possibility of rats with SCI being used as an experimental model of osteoporosis after SCI by examining the bone turnover status, bone mineral density and biomechanical competence of the bone. Also, pathogenesis mechanism of osteoporosis due to SCI was investigated. Method: SCI model was established by cutting off the distal thoracic spinal cord of rats; The investigation of biochemical values relating to bone resorption and formation, bone mineral density and biomechanics were performed; Ultrastructural investigation of bone were performed; BMP, IL-6 and TNF- a proteins in the bone were examined by using immunochemical stain combined with the imaging analysis techniques. Results: Calculus of the bladder was found in the SCI animals 6 week after injuries. Biochemical date demonstrated serum calcium, urine calcium, and urine calcium/creatinine rose dramatically compared with control groups at week 1,3,6.
(PO.05, 0.01, 0.05; PO.001, 0.001, 0.01; PO.05, 0.01, 0.01, respectively). Serum total alkaline phosphates dropped significantly at week 1 (P<0.01), and then returned to normal level at week 3,6. Bone mineral density measured by dual energy x-ray absorption at proximal of tibia, distal and proximal of femur showed a significant fall(P<0.05, 0.01, 0.05). There are significant decrease in value of maximum load, energy absorption capacity and structural rigidity in femoral neck through bending test after 6 weeks (P<0. 01, 0.05, 0.05). Ultrastructural investigation revealed little endoplasmic reticulum and Golgi apparatus were found in osteoblast and vacuole changes of mitochondrion indicating the low activity of osteoblast. Also, condensation of heterochromatin collected under the edge of nuclear membrane indicating the apoptosis were also found in osteoblasts. No differences were found between the SCI and control group in osteoblast ultrastructure at week 3. There were four basic changes in ultrastructure of osteocytes as follows. First, osteocytic dissolve characterized by decreased organelles. enlargement of bone lacuna, increasing concentrated wadding material and forming of osmium -favored stratum. Second, degenerative osteocytes revealed decreased organelles. sake-like change and some cells without integrated wall. Third, some typical necrosis cell showed nuclear pycnosis and cell collapsed. Fourth, changes such as bubble and bud. heterochromatin condensation, edge collection of heterochromatin under the nuclear membrane suggested apoptotic osteocytes. The expression of tumor necrosis factor-a (TNF-a) rose at week 1 significantly and returned to normal at week 6. Immunochemistry stain of bone morphogenetic protein (BMP) and interleukin-6 (IL-6) were more intensive in SCI groups at week 6 than that in control groups (PO.05, 0.05,respectfully), however, no difference was found between two groups at week 1. Conclusions: Osteoporosis develops at week 6 in rats suffering from SCI. Rats underwent SCI have a similar changes in bone turnover, bone loss site and the risk of fracture, so could be used as the animal model for osteoporosis after SCI. Decreased vigor and increased possibility of fall of total live osteoblasts resulted from
the apoptosis may contributed to the decreased bone forming ability in early state. Dissolve, degeneration, necrosis and apoptosis of osteocytes are the probable causes for decoupling. The data collected from the alteration of the immunochemistry indicated that cytokine play
引文
1 Vaziri ND, Pandian MR, Segal JL et al. Vitamin D, parathormone, and calcitonin profiles in persons with long standing spinal cord injury Arch Phys Med Rehabil.l994． 75:766-769．
2 Pietshmann P, Pils P, Woloszczuk W et al. Increased serum osteocalcin levels in patients with paraplegia. Paraplegia. 1992． 30:204-209．
3 Mechanick JI. Pomerantz F. Flanagan S.et al. Parathyroid hormone suppression in spinal cord injury patients is associated with the degree of neurologic impairment and not the level of injury Arch Phys Med Rehabil. 1997． 78:692-696．
4 Claus-Walker J, Halstead LS. Metabolic and endocrine changes in spinal cord injury: IV. compounded neurologic dysfunctions. Arch Phys Med Rehabil. 1982． 63:632-638．
5 Roberts-D,Lee-W,Cuneo-RC et al. Longitudinal study of bone turnover after acute spinal cord injury. J-Clin-Endocrinol-Metab. 1998 .83: 415-422．
6 Biering-Sorensen-F,Bohr-HH, Schaadt-OP. Longitudinal study of bone mineral content in the lumbar spine, the forearm and the lower extremities after spinal cord
injury. Eur-J-Clin-Invest. 1990． 20: 330-335．
7 Chantraine-A,Nusgens-B, Lapiere-CM. Bone remodeling during the development of osteoporosis in paraplegia. Calcif-Tissue-Int. 1986． 38: 323-327．
8 Kannisto-M, Alaranta-H, Merikanto-J, et al. Bone mineral status after pediatric spinal cord injury. Spinal-Cord. 1998 .9: 641-646．
9 Demirel-G, Yilmaz-H. Paker-N, et al. Osteoporosis after spinal cord injury Spinal-Cord. 1998 .12: 822-825．
10 Leslie-WD, Nance-PW. Dissociated hip and spine demineralization: a specific finding in spinal cord injury. Arch-Phys-Med-Rehabil. 1993． 74: 960-964．
11 Saltzstein-RJ, Hardin-S,Hastings-J. Osteoporosis in spinal cord injury: using an index of mobility and its relationship to bone density. J-Am-Paraplegia-Soc. 1992． 15:232-234．
12 Uebelhart D,Hartmann D,Vuagnat H.et al. Early modifications of biological markers of bone metabolism in spinal cord injury patients. A priliminary study. Scand J Rehabil Med. 1994;26:197-202．
13 Claus-walker J.Scurry RE.Carter.et al.Steady-state hormonal secretion in traumatic quadriplegia. J Clin Endocrinol Metab 1977:44:530．
14 Leblanc AD.Schneider VS. Evans HJ. et al. Bone mineral loss and recovery after 17 weeks of bed rest. J Bone Min Res 1990． 5:843-850．
15 Pun KK, Lau P, Wong FHW,et al.25-Hydroxycholecalciferol and insulin-like growth factor 1 are determinants of serum concentrations of osteocalcin in elederly subjects with and without spinal fractures. Bone. 1990． 11:397-400．
16 Bailey AJ, Wotton SF, Sime TJ,et al. Biochemical changes in the collagen of human osteoporotic bone matrix. Connect Tissue Res. 1993． 29:119-132．
17 Demulder-A, Guns-M,Ismail-A. Increased osteoclast-like cells formation in long-term bone marrow cultures from patients with a spinal cord injury. Calcif-Tissue-Int. 1998 .63: 396-400．
18 Nance-PW, Schryvers-O, Leslie-W, et al. Intravenous pamidronate attenuates bone density loss after acute spinal cord injury. Arch-Phys-Med-Rehabil. 1999． 80: 243-251．
19 Leeds-EM,Klose-KJ,Ganz-W, et al. Bone mineral density after bicycle ergometry training. Arch-Phys-Med-Rehabil. 1990 .71: 207-209．
2 Pietshmann P, Pils P, Woloszczuk W et al. Increased serum osteocalcin levels in patients with paraplegia. Paraplegia. 1992． 30:204-209．
3 Mechanick JI. Pomerantz F. Flanagan S.et al. Parathyroid hormone suppression in spinal cord injury patients is associated with the degree of neurologic impairment and not the level of injury Arch Phys Med Rehabil. 1997． 78:692-696．
4 Claus-Walker J, Halstead LS. Metabolic and endocrine changes in spinal cord injury: IV. compounded neurologic dysfunctions. Arch Phys Med Rehabil. 1982． 63:632-638．
5 Roberts-D,Lee-W,Cuneo-RC et al. Longitudinal study of bone turnover after acute spinal cord injury. J-Clin-Endocrinol-Metab. 1998 .83: 415-422．
6 Biering-Sorensen-F,Bohr-HH, Schaadt-OP. Longitudinal study of bone mineral content in the lumbar spine, the forearm and the lower extremities after spinal cord
injury. Eur-J-Clin-Invest. 1990． 20: 330-335．
7 Chantraine-A,Nusgens-B, Lapiere-CM. Bone remodeling during the development of osteoporosis in paraplegia. Calcif-Tissue-Int. 1986． 38: 323-327．
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