甲状旁腺激素促成骨作用的初步研究
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摘要
目的 探讨卵巢切除及间歇注射人甲状旁腺激素1-34片段对大鼠局部骨组织中胰岛素样生长因子1(IGF-1)表达的影响，同时检测血清中转化生长因子β_1(TGF-β_1)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)表达变化，从而评价各种细胞因子在绝经后骨质疏松的发病及PTH促成骨机理中的作用。
方法 3月龄雌性SD大鼠随机分为3组：假手术组(Sham)、去卵巢对照组(OVX)、去卵巢PTH注射组(PTH)，每组6只。假手术组暴露双侧卵巢并切除一块与卵巢大小相同的脂肪组织，去卵巢对照组和去卵巢PTH注射组于卵巢切除两周后分别皮下注射PBS溶剂或hPTH_(1-34)，6ug／100g体重，10天后所有大鼠一批处死，腹主动脉取血分离血清，-20℃保存。取胫骨中上1／3制备脱钙骨标本，免疫组化法(IHC)检测骨组织中细胞因子IGF-1的蛋白表达；酶联免疫吸附法(ELISA)检测血清中细胞因子TGF-β、IL-6、IL-1β、TNF-α的含量。
结果 成骨细胞呈线状排列于骨小梁的边缘，细胞因子IGF-1表达于成骨细胞胞浆，骨细胞、软骨细胞及骨基质中也存在IGF-1。不同部位的成骨细胞IGF-1的表达量并不一致，卵巢切除及PTH注射对不同部位成骨细胞IGF-1表达的影响也不一样。卵巢切除后软骨细胞
南京医科大学硕士学位论文
工GF一1表达降低，皮质骨及小梁骨部位成骨细胞的工GF一1表达无明显
变化。PTH注射对软骨区及皮质骨区IGF一表达无显著影响，但可以
明显增加小梁骨部位成骨细胞IGF一1的表达。萝尸巢切除后血清中细胞
因子TGF一日:升高，但去卵巢PTH注射组与去卵巢对照组无显著差异。
大鼠血清中IL一1日、工L6、TNF一a含量较低，各组间未见显著差异。
结论局部骨中细胞因子IGF一1在PTH的促成骨机理中起着重要
作用;间隙性PTH注射对血液循环中TGF一日，、IL一6、IL一1日、TNF-
a无明显影响，其在局部骨组织中的变化尚有待于进一步研究。
Objective: To evaluate the effects of ovariectomy and intermittent injection of human parathyroid hormone(1-34) on the protein level of local insulin-like growth factor-1 (IGF-1) , Meanwhile transforming growth factor- P 1 (TGF- P 1), interlukin-6(IL-6), interlukin-1 P (IL-1 P ), tumor necrosis factor- a (TNF- a ) in serum were detected.
Methods: Three month-old rats were ovariectomied(OVX) or sham-operated (SHAM). Two weeks after operation, OVX rats received daily subcutaneous injection of human parathyroid hormone fragment 1-34 (hPTH 1-34), 6ug/100g body weight or vehicle for 10 days. Then all animals were sacrificed. The tibiae of the rats were kept for the samples of decalcification, IGF-1 in bone tissue was measured by immunohistochemistry and Enzyme linked-immuno-sorbent assay (ELISA) was used to detect the TGF- P 1, IL-6, IL-1 P and TNF- a in serum.
Results: IGF-1 locates in the cytoplasts of osteoblasts, OVX has no effects on the protein level of IGF-1 in rat bone tissue and injection of hPTH (1-34) can increase its expression in cancellous , However no
difference of TGF- β1 level was found in OVX rats treated with or without hPTH (1-34) . The concentrations of IL-1 β, IL-6, TNF- are lower and no difference were found in three groups.
Conclusion: Our findings suggest that IGF-1 in local bone tissue plays an important role in the anabolic effects of PTH and intermittent injection of PTH has no effects on the concentration of TGF- β1 , IL-6, IL-1 β , TNF- in serum.
方法 3月龄雌性SD大鼠随机分为3组：假手术组(Sham)、去卵巢对照组(OVX)、去卵巢PTH注射组(PTH)，每组6只。假手术组暴露双侧卵巢并切除一块与卵巢大小相同的脂肪组织，去卵巢对照组和去卵巢PTH注射组于卵巢切除两周后分别皮下注射PBS溶剂或hPTH_(1-34)，6ug／100g体重，10天后所有大鼠一批处死，腹主动脉取血分离血清，-20℃保存。取胫骨中上1／3制备脱钙骨标本，免疫组化法(IHC)检测骨组织中细胞因子IGF-1的蛋白表达；酶联免疫吸附法(ELISA)检测血清中细胞因子TGF-β、IL-6、IL-1β、TNF-α的含量。
结果 成骨细胞呈线状排列于骨小梁的边缘，细胞因子IGF-1表达于成骨细胞胞浆，骨细胞、软骨细胞及骨基质中也存在IGF-1。不同部位的成骨细胞IGF-1的表达量并不一致，卵巢切除及PTH注射对不同部位成骨细胞IGF-1表达的影响也不一样。卵巢切除后软骨细胞
南京医科大学硕士学位论文
工GF一1表达降低，皮质骨及小梁骨部位成骨细胞的工GF一1表达无明显
变化。PTH注射对软骨区及皮质骨区IGF一表达无显著影响，但可以
明显增加小梁骨部位成骨细胞IGF一1的表达。萝尸巢切除后血清中细胞
因子TGF一日:升高，但去卵巢PTH注射组与去卵巢对照组无显著差异。
大鼠血清中IL一1日、工L6、TNF一a含量较低，各组间未见显著差异。
结论局部骨中细胞因子IGF一1在PTH的促成骨机理中起着重要
作用;间隙性PTH注射对血液循环中TGF一日，、IL一6、IL一1日、TNF-
a无明显影响，其在局部骨组织中的变化尚有待于进一步研究。
Objective: To evaluate the effects of ovariectomy and intermittent injection of human parathyroid hormone(1-34) on the protein level of local insulin-like growth factor-1 (IGF-1) , Meanwhile transforming growth factor- P 1 (TGF- P 1), interlukin-6(IL-6), interlukin-1 P (IL-1 P ), tumor necrosis factor- a (TNF- a ) in serum were detected.
Methods: Three month-old rats were ovariectomied(OVX) or sham-operated (SHAM). Two weeks after operation, OVX rats received daily subcutaneous injection of human parathyroid hormone fragment 1-34 (hPTH 1-34), 6ug/100g body weight or vehicle for 10 days. Then all animals were sacrificed. The tibiae of the rats were kept for the samples of decalcification, IGF-1 in bone tissue was measured by immunohistochemistry and Enzyme linked-immuno-sorbent assay (ELISA) was used to detect the TGF- P 1, IL-6, IL-1 P and TNF- a in serum.
Results: IGF-1 locates in the cytoplasts of osteoblasts, OVX has no effects on the protein level of IGF-1 in rat bone tissue and injection of hPTH (1-34) can increase its expression in cancellous , However no
difference of TGF- β1 level was found in OVX rats treated with or without hPTH (1-34) . The concentrations of IL-1 β, IL-6, TNF- are lower and no difference were found in three groups.
Conclusion: Our findings suggest that IGF-1 in local bone tissue plays an important role in the anabolic effects of PTH and intermittent injection of PTH has no effects on the concentration of TGF- β1 , IL-6, IL-1 β , TNF- in serum.
引文
1. Genant HK, Cooper C, Poor G, et al. Interim report and recommendations of the world health organization task-force for
osteoporosis. Osteoporos Int, 1999, 10: 259-264.
2. Effects of hormone therapy on bone mineral densit: results from the postmenopausal estrogen/progestin interventions (PEPI) trial. The Writing Group for the PEPI. JAMA, 1996, 276:1389-96.
3. Rubin MR, Cosman F, Lindsay R, et al. The anabolic effects of parathyroid hormone. Osteoporos Int, 2002, 13: 267-277.
4. Zhang KQ, Chen JW, Li QN, et al. Effect of intermittent injection of recombinant human parathyroid hormone on bone histomorphometry of ovariectomized rats. Acta Pharmacol Sin, 2002, 23(7): 659-662.
5. Neer RM, Arnaud CD, Zanchetta JR, et al. Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med, 2001, 344:1434-1441.
6. Watson P, Lazowski D, Han V, et al. Parathyroid hormone restores bone mass and enhances osteoblast insulin-like growth factorl gene expression in ovariectomized rats. Bone, 1995, 16(3): 357-365.
7. Bikle DD, Sakata T, Leary C, et al. Insulin-like growth factor 1 is required for the anabolic actions of parathyroid hormone on mouse bone. J Bone Miner Res, 2002, 17(9): 1570-1578.
8. Baylink DJ, Finkelman RD, Mohan S. Growth factors to stimulate bone formation. J Bone Miner Res. 1993, 8:S2; S565-S572.
9. Farley JR, Stilt-Coffing B. Apotptosis may determine the release of
skeletal alkaline phospthatase activity from human osteoblast-line cells. CalcifTissue Int, 2001,68(1):43-52.
10. Ostrowska Z, Kos-Kudla B, Marek B, et al. Dynamic pattern of IGF-1 and chosen biochemical markers of bone metabolism in a rat model of postmenopausal osteoporosis. Endocri Regul, 2002, 36:9-7.
11. Suliman IA, EI-Bakri NK, Adem A, et al. The effect of ovariectomy and ovarian steroid treatment on growth hormone and insulin-like growth factor-I levers in the rat femur. J orthopaedic res, 2001, 19: 1008-1012.
12. Goldring, Steven R, Goldring, et al. Cytokines and Skeletal Physiology [J]. Papers From The International Symposium On Bone And Soft Tissue Allografts, 1996,324:13-23.
13. Zheng SX, Vrindts Y, Lopez M, et al. Increase in cytokine production (IL-1 beta, IL-6, TNF-alpha but not IFN-gamma, GM-CSF or LIF) by stimulated whole blood cells in postmenopausal osteoporosi. Maturitas, 1997, 26(1): 63-71.
14. Ohmori S, Kanda K, Kawano S, et al. Effects of estrogen on tail suspension-induced disuse atrophy in ovariectomized rats: evaluation of the expression of interleukin-6 mRNA in the femur. Environmental medicine: annual report of the research institute of environmental medicine, Nagoya university, 2001, 45(1): 12-14.
15. Brooks-Asplund EM, Tupper CE, Daun JM, et al. Hormonal
modulation of interleukin-6, tumor necrosis factor and associated receptor secretion in postmenopausal women. Cytokine, 2002, 19(4): 193-200.
16. Baylink D J, Finkelman RD, Mohan S. Growth factors to stimulate bone formation. J Bone Miner Res. 1993, 8:S2; S565-S572.
17．秦林林，陈金标，龚海洋，等。不同月龄雌性大鼠骨质疏松模型研究。中日友好医院学报。1997，11(1)：6-10．
18．席越，王戈平，黄啸原等．骨组织病理解剖学技术．人民卫生出版社，1997．
19．刘大维，初同伟，张良等．EDTA微波脱钙骨的免疫组化染色．第三军医大学学报，2003，25(1)：77-78
20. Wenhan C, Chialing T, Tsui-hua C, et al. Expression and signal transduction of calcium-sensing receptors in cartilage and bone. Endocrinol. 1999, 140(12): 5883-5893
21. McCarthy TL, Centrella M. Local IGF-1 expression and bone formation. Growth-Horm-IGF-Res, 2001, 11:213-219.
22．张克勤，陈家伟。甲状旁腺激素的促成骨作用研究。南京医科大学博士论文。
23. Locklin RM, Khosla S, Riggs BL. Mechanisms of biphasic anabolic and catabolic effects of parathyroid hormone(PTH) on bone cells. Bone, 2001,28: $80.
osteoporosis. Osteoporos Int, 1999, 10: 259-264.
2. Effects of hormone therapy on bone mineral densit: results from the postmenopausal estrogen/progestin interventions (PEPI) trial. The Writing Group for the PEPI. JAMA, 1996, 276:1389-96.
3. Rubin MR, Cosman F, Lindsay R, et al. The anabolic effects of parathyroid hormone. Osteoporos Int, 2002, 13: 267-277.
4. Zhang KQ, Chen JW, Li QN, et al. Effect of intermittent injection of recombinant human parathyroid hormone on bone histomorphometry of ovariectomized rats. Acta Pharmacol Sin, 2002, 23(7): 659-662.
5. Neer RM, Arnaud CD, Zanchetta JR, et al. Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med, 2001, 344:1434-1441.
6. Watson P, Lazowski D, Han V, et al. Parathyroid hormone restores bone mass and enhances osteoblast insulin-like growth factorl gene expression in ovariectomized rats. Bone, 1995, 16(3): 357-365.
7. Bikle DD, Sakata T, Leary C, et al. Insulin-like growth factor 1 is required for the anabolic actions of parathyroid hormone on mouse bone. J Bone Miner Res, 2002, 17(9): 1570-1578.
8. Baylink DJ, Finkelman RD, Mohan S. Growth factors to stimulate bone formation. J Bone Miner Res. 1993, 8:S2; S565-S572.
9. Farley JR, Stilt-Coffing B. Apotptosis may determine the release of
skeletal alkaline phospthatase activity from human osteoblast-line cells. CalcifTissue Int, 2001,68(1):43-52.
10. Ostrowska Z, Kos-Kudla B, Marek B, et al. Dynamic pattern of IGF-1 and chosen biochemical markers of bone metabolism in a rat model of postmenopausal osteoporosis. Endocri Regul, 2002, 36:9-7.
11. Suliman IA, EI-Bakri NK, Adem A, et al. The effect of ovariectomy and ovarian steroid treatment on growth hormone and insulin-like growth factor-I levers in the rat femur. J orthopaedic res, 2001, 19: 1008-1012.
12. Goldring, Steven R, Goldring, et al. Cytokines and Skeletal Physiology [J]. Papers From The International Symposium On Bone And Soft Tissue Allografts, 1996,324:13-23.
13. Zheng SX, Vrindts Y, Lopez M, et al. Increase in cytokine production (IL-1 beta, IL-6, TNF-alpha but not IFN-gamma, GM-CSF or LIF) by stimulated whole blood cells in postmenopausal osteoporosi. Maturitas, 1997, 26(1): 63-71.
14. Ohmori S, Kanda K, Kawano S, et al. Effects of estrogen on tail suspension-induced disuse atrophy in ovariectomized rats: evaluation of the expression of interleukin-6 mRNA in the femur. Environmental medicine: annual report of the research institute of environmental medicine, Nagoya university, 2001, 45(1): 12-14.
15. Brooks-Asplund EM, Tupper CE, Daun JM, et al. Hormonal
modulation of interleukin-6, tumor necrosis factor and associated receptor secretion in postmenopausal women. Cytokine, 2002, 19(4): 193-200.
16. Baylink D J, Finkelman RD, Mohan S. Growth factors to stimulate bone formation. J Bone Miner Res. 1993, 8:S2; S565-S572.
17．秦林林，陈金标，龚海洋，等。不同月龄雌性大鼠骨质疏松模型研究。中日友好医院学报。1997，11(1)：6-10．
18．席越，王戈平，黄啸原等．骨组织病理解剖学技术．人民卫生出版社，1997．
19．刘大维，初同伟，张良等．EDTA微波脱钙骨的免疫组化染色．第三军医大学学报，2003，25(1)：77-78
20. Wenhan C, Chialing T, Tsui-hua C, et al. Expression and signal transduction of calcium-sensing receptors in cartilage and bone. Endocrinol. 1999, 140(12): 5883-5893
21. McCarthy TL, Centrella M. Local IGF-1 expression and bone formation. Growth-Horm-IGF-Res, 2001, 11:213-219.
22．张克勤，陈家伟。甲状旁腺激素的促成骨作用研究。南京医科大学博士论文。
23. Locklin RM, Khosla S, Riggs BL. Mechanisms of biphasic anabolic and catabolic effects of parathyroid hormone(PTH) on bone cells. Bone, 2001,28: $80.