转鲑鱼降钙素基因酵母对骨质疏松和血管钙化作用的研究
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摘要
降钙素(calcitonin,CT)是一种32个氨基酸组成的多肽类激素。临床应用鲑鱼降钙素(salmon calcitonin,sCT)治疗骨质疏松,而现有给药途径仅仅注射和鼻喷两种,且昂贵的价格也限制了它在我国的普及。实现鲑鱼降钙素的口服给药(转鲑鱼降钙素基因酵母yAGA2-sCT)将是最为理想的选择。
为研究转鲑鱼降钙素基因酵母(yAGA2-sCT)对骨质疏松大鼠的作用,我们建立了骨质疏松大鼠模型,观察sCT和yAGA2-sCT对骨质疏松大鼠骨密度、骨形态计量学影响;同时检测血钙、血碱性磷酸酶、血清骨钙素、血清胰岛素样生长因子-1以及尿钙、羟脯氨酸等经典的骨代谢生化标志物水平以观察sCT和yAGA2-sCT对骨代谢的影响。实验分假手术组、模型对照组、sCT组及yAGA2-sCT组。通过大鼠股骨X光照片、光镜观察及骨密度值检测说明骨质疏松大鼠造模成功。sCT组及yAGA2-sCT组的腰椎和股骨骨密度及骨代谢生化标志物水平明显得到改善,提示sCT及yAGA2-sCT通过抑制骨吸收从而抑制了骨质疏松。sCT组及yAGA2-sCT组两组比较差异无显著性,提示口服yAGA2-sCT与sCT注射液对骨质疏松大鼠有相似的作用,均具有干预骨重建、抑制骨质疏松的作用。
为进一步探讨转鲑鱼降钙素基因酵母(yAGA2-sCT)对骨质疏松的作用机制,我们观察了yAGA2-sCT对体外成骨细胞的作用。应用酶消化-连续组织块法体外培养大鼠成骨细胞,观察sCT和yAGA2-sCT对体外培养成骨细胞增殖、分化、矿化功能的影响。经病理学检查及染色证实体外培养成骨细胞成功;sCT及yAGA2-sCT对体外培养大鼠成骨细胞的增殖、分化和矿化功能具有刺激作用。
众所周知老年患者发生骨质疏松的同时常常伴发血管钙化。近来认为三个新发现的肿瘤坏死因子家族成员骨保护素(Osteoprotegrin,OPG),核因子kB受体激活物配体( Receptor activator of nuclear factor kappa B ligand,RANKL)和核因子kB受体激活物(Receptor activator of nuclear factor kappaB,RANK),亦称OPG/RANKL/RANK系统,参与骨质疏松和血管钙化的调节过程,是这两种伴发疾病之间的分子联系。
为研究OPG/RANKL/RANK系统在血管钙化中的调节作用以及降钙素对OPG/RANKL/RANK途径的干预作用,本实验建立了大鼠动脉钙化模型,病理学观察血管钙化情况,测定血管组织中钙含量及碱性磷酸酶活性,免疫组化法检测OPG和RANKL在血管组织中的表达。实验分对照组、模型组、sCT组及yAGA2-sCT组。病理学观察成功建立维生素D3诱发的血管钙化模型;与对照组比较,钙化血管组织中钙含量及碱性磷酸酶活性增高;模型组血管壁OPG表达明显减少而RANKL表达明显增加。与模型组比较,sCT组和yAGA2-sCT组血管钙化程度明显减轻,血管壁OPG表达明显增加而RANKL表达明显减少。提示OPG/RANKL/RANK系统参与血管钙化的调节,sCT和yAGA2-sCT可通过OPG/RANKL/RANK途径有效抑制血管钙化。
为进一步观察RANKL对大鼠血管平滑肌细胞(vascular smooth muscle cell,VSMC)体外钙化的调节作用及降钙素对其调节作用的影响,我们体外培养大鼠VSMC,建立体外VSMC钙化模型,在培养介质中分别加入RANKL及降钙素,观察细胞钙沉积及细胞碱性磷酸酶活性、骨钙素的变化情况。VSMC经von Kossa染色证实动脉钙化模型成功建立。实验分对照组、钙化模型组、RANKL组、sCT组及yAGA2-sCT组。钙化模型组、RANKL组、sCT组及sCT组钙沉积、碱性磷酸酶活性、骨钙素水平明显高于对照组;与钙化模型组比较,RANKL组钙沉积、碱性磷酸酶活性、骨钙素水平均明显增高;与RANKL组比较, sCT组和yAGA2-sCT组钙沉积、碱性磷酸酶活性、骨钙素水平明显降低。提示RANKL促进钙化培养的VSMC钙沉积、碱性磷酸酶活性及骨钙素的表达,而sCT和yAGA2-sCT能够抑制RANKL的以上效应。此结果进一步提示RANKL可促进钙化培养的VSMC的钙质沉积,其作用可能与增加细胞碱性磷酸酶活性及骨钙素的表达、促进VSMC向成骨细胞转化有关,而sCT和yAGA2-sCT可通过对RANKL的抑制作用、通过OPG/RANKL/RANK途径抑制VSMC钙化。
Calcitonin is a 32-amino-acid-long peptide . Salmon calcitonin (sCT) is used to treat osteoporosis by injection or nasal administration, and the high price is the other limitation of sCT. To study the oral delivery of salmon calcitonin and to low the price of it, yeast yAGA2-sCT will be the best choice.
To observe the therapeutic effect of yAGA2-sCT on osteoporosis in rat, at first we established a rat model of osteoporosis. Then we determined bone mineral density(BMD) and examined calcium(Ca),alkaline phosphatase(AKP),bone gla protein(BGP), insulin-like growth factor-1(IGF-1) in serum and Ca, hydroxyproline(HOP) in urine. A rat osteoporosis model induced by ovariectomy was established successfully, which was verified by X-ray, BMD and histological examination. When sCT and yAGA2-sCT were added, both the BMD and biochemical markers of bone metabolism improved significantly. There is no difference between the two groups of sCT and yAGA2-sCT. The result suggest that the bone resorption and bone loss of osteoporosis in rat can be prevented by sCT and yAGA2-sCT.
To study the mechanism of yAGA2-sCT to osteoporosis, the osteoblasts of newborn SD rats were obtained through the enzymatic digestion- consecutive tissue method. sCT and yAGA2-sCT were added into the culture medium, and the proliferation, differentiation and mineralization of the osteoblasts were observed. The result showed us that the in vitro osteoblasts were successfully cultured , which was verified histologically. From the result we can conclud that both sCT and yAGA2-sCT can stimulate formation of bone through affecting the proliferation , differentiation and mineralization of osteoblasts in vitro .
Osteoporosis is usually occur concomitantly with arterial calcification in elderly. Recent studies find that OPG/RANKL/RANK, newly defined members of TNF super-family of receptors, participate in regulation of osteoporosis and arterial calcification, and are the connection of these two disease process.
To study the regulatory role of OPG/RANKL/RANK system in arterial calcification and the effect of calcitonin on OPG/RANKL/RANK system, at first we established a rat model of arterial calcification. Then we examined calcium and alkaline phosphatase (AKP) level in arterial tissue and determined the expression of OPG/RANKL in arterial tissue by immunohistochemistry. A rat arterial calcification model induced by Vitamin D3 was established successfully, which was verified by examining the arteries histologically. The calcium and alkaline phosphatase (AKP) level in arterial tissue of the rat model were higher than that of control group. The expression of OPG was prominently lower and RANKL was prominently higher in the arterial tissue of rat model than that of control. When sCT and yAGA2-sCT were added, the OPG expression was markedly increased and RANKL level was obviously decreased in arterial tissue of the rat than that of rat model without calcitonin. The result suggest that OPG/RANKL participates the regulation of arterial calcification, sCT and yAGA2-sCT effectively inhibit arterial calcification through OPG/RANKL/RANK system.
To investigate the regulation effect of RANKL on in vitro calcification of rat aortic vascular smooth muscle cells (VSMC) and the role of calcitonin in this effect,we cultured rat aortic VSMC in vitro and established a calcification model with the cells. RANKL and CT were added into the culture media, then the calcium deposition, alkaline phosphatase (AKP) activity, and alterations of expression of osteocalcin were examined. Von Kossa staining VSMC demonstrate the in vitro calcification model was successfully established. The Calcium content, AKP activity and the expression of osteocalcin were markedly increased in calcification, RANKL , sCT and yAGA2-sCT group than that of control group. But they were obviously decreased in sCT and yAGA2-sCT group than that of RANKL group, which suggest that RANKL increase the calcium deposition, AKP activity, and expression of osteocalcin in VSMC, whereas sCT and yAGA2-sCT inhibit this effect. RANKL increase calcium deposition in VSMC, possibly through increasing AKP activity and expression of osteocalcium and thus promote the transformation of VSMC to osteoblast. sCT and yAGA2-sCT may inhibit VSMC calcification through OPG/RANKL/RANK system.
为研究转鲑鱼降钙素基因酵母(yAGA2-sCT)对骨质疏松大鼠的作用,我们建立了骨质疏松大鼠模型,观察sCT和yAGA2-sCT对骨质疏松大鼠骨密度、骨形态计量学影响;同时检测血钙、血碱性磷酸酶、血清骨钙素、血清胰岛素样生长因子-1以及尿钙、羟脯氨酸等经典的骨代谢生化标志物水平以观察sCT和yAGA2-sCT对骨代谢的影响。实验分假手术组、模型对照组、sCT组及yAGA2-sCT组。通过大鼠股骨X光照片、光镜观察及骨密度值检测说明骨质疏松大鼠造模成功。sCT组及yAGA2-sCT组的腰椎和股骨骨密度及骨代谢生化标志物水平明显得到改善,提示sCT及yAGA2-sCT通过抑制骨吸收从而抑制了骨质疏松。sCT组及yAGA2-sCT组两组比较差异无显著性,提示口服yAGA2-sCT与sCT注射液对骨质疏松大鼠有相似的作用,均具有干预骨重建、抑制骨质疏松的作用。
为进一步探讨转鲑鱼降钙素基因酵母(yAGA2-sCT)对骨质疏松的作用机制,我们观察了yAGA2-sCT对体外成骨细胞的作用。应用酶消化-连续组织块法体外培养大鼠成骨细胞,观察sCT和yAGA2-sCT对体外培养成骨细胞增殖、分化、矿化功能的影响。经病理学检查及染色证实体外培养成骨细胞成功;sCT及yAGA2-sCT对体外培养大鼠成骨细胞的增殖、分化和矿化功能具有刺激作用。
众所周知老年患者发生骨质疏松的同时常常伴发血管钙化。近来认为三个新发现的肿瘤坏死因子家族成员骨保护素(Osteoprotegrin,OPG),核因子kB受体激活物配体( Receptor activator of nuclear factor kappa B ligand,RANKL)和核因子kB受体激活物(Receptor activator of nuclear factor kappaB,RANK),亦称OPG/RANKL/RANK系统,参与骨质疏松和血管钙化的调节过程,是这两种伴发疾病之间的分子联系。
为研究OPG/RANKL/RANK系统在血管钙化中的调节作用以及降钙素对OPG/RANKL/RANK途径的干预作用,本实验建立了大鼠动脉钙化模型,病理学观察血管钙化情况,测定血管组织中钙含量及碱性磷酸酶活性,免疫组化法检测OPG和RANKL在血管组织中的表达。实验分对照组、模型组、sCT组及yAGA2-sCT组。病理学观察成功建立维生素D3诱发的血管钙化模型;与对照组比较,钙化血管组织中钙含量及碱性磷酸酶活性增高;模型组血管壁OPG表达明显减少而RANKL表达明显增加。与模型组比较,sCT组和yAGA2-sCT组血管钙化程度明显减轻,血管壁OPG表达明显增加而RANKL表达明显减少。提示OPG/RANKL/RANK系统参与血管钙化的调节,sCT和yAGA2-sCT可通过OPG/RANKL/RANK途径有效抑制血管钙化。
为进一步观察RANKL对大鼠血管平滑肌细胞(vascular smooth muscle cell,VSMC)体外钙化的调节作用及降钙素对其调节作用的影响,我们体外培养大鼠VSMC,建立体外VSMC钙化模型,在培养介质中分别加入RANKL及降钙素,观察细胞钙沉积及细胞碱性磷酸酶活性、骨钙素的变化情况。VSMC经von Kossa染色证实动脉钙化模型成功建立。实验分对照组、钙化模型组、RANKL组、sCT组及yAGA2-sCT组。钙化模型组、RANKL组、sCT组及sCT组钙沉积、碱性磷酸酶活性、骨钙素水平明显高于对照组;与钙化模型组比较,RANKL组钙沉积、碱性磷酸酶活性、骨钙素水平均明显增高;与RANKL组比较, sCT组和yAGA2-sCT组钙沉积、碱性磷酸酶活性、骨钙素水平明显降低。提示RANKL促进钙化培养的VSMC钙沉积、碱性磷酸酶活性及骨钙素的表达,而sCT和yAGA2-sCT能够抑制RANKL的以上效应。此结果进一步提示RANKL可促进钙化培养的VSMC的钙质沉积,其作用可能与增加细胞碱性磷酸酶活性及骨钙素的表达、促进VSMC向成骨细胞转化有关,而sCT和yAGA2-sCT可通过对RANKL的抑制作用、通过OPG/RANKL/RANK途径抑制VSMC钙化。
Calcitonin is a 32-amino-acid-long peptide . Salmon calcitonin (sCT) is used to treat osteoporosis by injection or nasal administration, and the high price is the other limitation of sCT. To study the oral delivery of salmon calcitonin and to low the price of it, yeast yAGA2-sCT will be the best choice.
To observe the therapeutic effect of yAGA2-sCT on osteoporosis in rat, at first we established a rat model of osteoporosis. Then we determined bone mineral density(BMD) and examined calcium(Ca),alkaline phosphatase(AKP),bone gla protein(BGP), insulin-like growth factor-1(IGF-1) in serum and Ca, hydroxyproline(HOP) in urine. A rat osteoporosis model induced by ovariectomy was established successfully, which was verified by X-ray, BMD and histological examination. When sCT and yAGA2-sCT were added, both the BMD and biochemical markers of bone metabolism improved significantly. There is no difference between the two groups of sCT and yAGA2-sCT. The result suggest that the bone resorption and bone loss of osteoporosis in rat can be prevented by sCT and yAGA2-sCT.
To study the mechanism of yAGA2-sCT to osteoporosis, the osteoblasts of newborn SD rats were obtained through the enzymatic digestion- consecutive tissue method. sCT and yAGA2-sCT were added into the culture medium, and the proliferation, differentiation and mineralization of the osteoblasts were observed. The result showed us that the in vitro osteoblasts were successfully cultured , which was verified histologically. From the result we can conclud that both sCT and yAGA2-sCT can stimulate formation of bone through affecting the proliferation , differentiation and mineralization of osteoblasts in vitro .
Osteoporosis is usually occur concomitantly with arterial calcification in elderly. Recent studies find that OPG/RANKL/RANK, newly defined members of TNF super-family of receptors, participate in regulation of osteoporosis and arterial calcification, and are the connection of these two disease process.
To study the regulatory role of OPG/RANKL/RANK system in arterial calcification and the effect of calcitonin on OPG/RANKL/RANK system, at first we established a rat model of arterial calcification. Then we examined calcium and alkaline phosphatase (AKP) level in arterial tissue and determined the expression of OPG/RANKL in arterial tissue by immunohistochemistry. A rat arterial calcification model induced by Vitamin D3 was established successfully, which was verified by examining the arteries histologically. The calcium and alkaline phosphatase (AKP) level in arterial tissue of the rat model were higher than that of control group. The expression of OPG was prominently lower and RANKL was prominently higher in the arterial tissue of rat model than that of control. When sCT and yAGA2-sCT were added, the OPG expression was markedly increased and RANKL level was obviously decreased in arterial tissue of the rat than that of rat model without calcitonin. The result suggest that OPG/RANKL participates the regulation of arterial calcification, sCT and yAGA2-sCT effectively inhibit arterial calcification through OPG/RANKL/RANK system.
To investigate the regulation effect of RANKL on in vitro calcification of rat aortic vascular smooth muscle cells (VSMC) and the role of calcitonin in this effect,we cultured rat aortic VSMC in vitro and established a calcification model with the cells. RANKL and CT were added into the culture media, then the calcium deposition, alkaline phosphatase (AKP) activity, and alterations of expression of osteocalcin were examined. Von Kossa staining VSMC demonstrate the in vitro calcification model was successfully established. The Calcium content, AKP activity and the expression of osteocalcin were markedly increased in calcification, RANKL , sCT and yAGA2-sCT group than that of control group. But they were obviously decreased in sCT and yAGA2-sCT group than that of RANKL group, which suggest that RANKL increase the calcium deposition, AKP activity, and expression of osteocalcin in VSMC, whereas sCT and yAGA2-sCT inhibit this effect. RANKL increase calcium deposition in VSMC, possibly through increasing AKP activity and expression of osteocalcium and thus promote the transformation of VSMC to osteoblast. sCT and yAGA2-sCT may inhibit VSMC calcification through OPG/RANKL/RANK system.
引文
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Khosla S. Minireview: The OPG/RANKL/RANK System. Endocrinology. 2001, 142(12): 5050-5055.
Kiel, Kauppila, Cupples, et a1. Bone loss and the progression of abdominal aortic calcification over a 25 year period: the Framingham Heart Study. Calcif Tissue Int. 2001,68: 27l-276.
Kruger MC, Horrobin DF. Calcium metabolism, osteoporosis and essential fatty acids: a review. Prog Lipid Res. 1997, 36(2-3): 131-l51.
Layne JE, Nelson ME. The effects of progressive resistance training on bone density: a review. Med Sci Sports Exerc. 1999, 31 (1): 25-30.
Lee Co, Herrmann RG. Efect of Vitamin D, Sucrose, Corn Oil and Endocrines on Tissue Cholesterol in Rats. Circ Res. l959, 7(3): 354-358.
Lorenz, Shui, Riggs, et a1. Effects of immunosuppressants on receptor activator of NF-kappa B ligand and osteoprotegerin production by human osteoblastic and coronary artery smooth muscle cells. Biochem Biophys Res Commun. 2001,280: 334-339.
Luo G, Ducy P, McKee MD, et a1. Spontaneous calcification of arteries and cartilage in mice lacking matrix Gla protein. Nature.1997,386: 78-81.
Malyankar UM, Scatena M, Suchland KL, et al. Osteoprotegerin is An Alpha v beta 3-induced, NF-kappa B-dependent Survival Factor For Endothelial cells. J Biol Chem. 2000, 275: 20959-20962.
Mancini N, Moradi-Bidhendi ML, Brandi, et al. Modulation of the effects of osteoprotegerin (OPG) ligand in a human leukemic cell line by OPG and calcitonin. Biochemical and Biophysical Research Communications. 2000, 279: 391-397.
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Min H, Morony S, Sarosi I, et al. Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis. Exp Med. 2000, 192: 463-474.
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Oei HH, Wliegenthart R, Hak AE, et al. The Association Between Coronary Calcification Assessed by Electron Beam Computed Tomography and Measures of Extracoronary Atherosclerosis:The Rotterdam Coronary Calcification study. Am Coll Cardiol. 2002, 39(11): 1745-1751
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Pak Cyc, Sakhaee K, Adams-Huet B, et al. Treatment of postmenopausal osteoporosis with slow-release sodium fluoride: final update of a randomized controlled trial. Ann Lntern Med, 1995,123: 401.
Pak CYC, Sakhaee K, Beu NH, et al. Comparison of nonrandomized trials with slow-release sodium fluoride with a randomized placebo controlled trial in postmenopausal osteoporosis. Bone Min Res. 1996, 11: 160.
Parhami, Garfinkel, Linda. Role of lipids in osteoporosis. Arterioscler Thromb Vase Biol. 2000,20: 2346-2348.
Peck WA, Birge ST. Bone cells: biochemical and biological studies after enzymatic isolation . Science. 1964, 146: 1746-1750.
Price PA,Fause SA, Williamson MK, et a1.Bisphosphonates Alendronate and Ibandronate Inhibit Artery Calcification at Doses Comparable to Those That Inhibit Bone Resorption. Arterioscler Thmmb Vasc Biol. 2001, 21(5): 817-824.
Price PA, June HH, Buckley JR, et al. Osteoprotegerin Inhibits Artery Calcification Induced by Warfarin and by Vitamin D. Arterioscler Thromb Vasc Biol .2001,21: 1610-1616.
Proudfoot D, Skepper JN, Shanahan CM, et a1. Calcification of human vascular cells in vitro is correlated with high levels of matrix Gla protein and low levels of osteopontin expression. Arterioscler Thromb Vasc Biol. 1998,18: 379-388
Proudfoot D, Skepper JN, Hegyi L, et a1. The role of apoptosis in the initiation of vascular calcification. Z Kardio1. 2001, 90(Suppl 3): 43-46
Ringe JD, Meunier PJ. What is the future for fluoride in the treatment of osteoporosis. Osteoporosis Int . 1995,5: 71.
Rood man GD. Cell biology of the osetoclast. Experimental Hematology. 1999,27:1229-1241.
Schoppet, Preissner, Hofbauer, et a1. RANK ligand and osteoprotegerin: paracrine regulators or bone metabolism and vascular function.Arterioscler Thtomb Vasc Biol. 2002, 22: 549-553.
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Shanahan GM, Cary NY, Metcalfe JC, et al. High expression of genes for calcification-regulating proteins in human atherosclerotic plaques. J Clin Invest.1994, 93: 2393-2402.
Shioi A, Nishizawa Y, Jono S, et a1.β-Glycerophosphate accelerates calcification in cultured bovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol.1995,15: 2003-2009.
Shearer MJ.Role of vitamin K and Gla proteins in the pathophysiology of osteoporosis and vascular calcification. Curt Opin Clin Nutr Metab Care. 2000, 3: 433-438.
Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell. 1997,89:309-319.
Stein GS, Lian JB, Stein JL, et al. Transcroptional control of osteoblast growth and differentiation Physilolgical. Reviews. 1996, 76 (2): 593-627.
Sugihara N, Matsuzaki. The influence of severe bone loss on mitral annular calcification in postmenopausal osteoporosis of elderly Japfnese wonen. Jpn Circ J. l993, 57: 14-26.
Sun PN, Zhang XC, Zang XN, Zhou XJ, et al. The anti-hypercalcemic effect of orally administered recombinant Saccharomyces cerevisiae expressing salmon calcitonin on hypercalcemic rats. Biotechnol Lett. 2007,29(7): 1013-1018.
Tsuda E, Goto M, Mochizuki S, et a1. Isolation of a novel cytokine form human fibroblasts that specifically inhibits 0steoclast0genesis. Bioche Biophys Res Commun.1997,234: 137-142.
Whitfield J, Morley P, Willick G. The parathyroid hormone, its fragments and analogues potent bone-builders for treating osteoporosis. Expert Opin lnvestig Drugs.2000,9(6): 1293-1235.
Wada T, McKee MD, Steitz S, et a1. Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin. Circ Res. 1999, 84: 166-178
Wallin R, Wajih N, Greenwood GT, et a1. Arterial calcification:a review of mechanisms, animal models, and the prospects for therapy . Med Res Rev.2001,21: 274-301.
Wong GL, Cohn DV. Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to poly meric surfaces. Pro Natl Acad Sci USA. 1975, 72: 3167-3171.
Ylitalo R. Bisphosphonates and atherosclerosis. Gen Pharmaeol. 2000, 35: 287-296.
Zaidi M, Inzerillo AM, Moonga BS, et al. Forty Years of Calcitonin-Where Are We Now? A Tribute to the Work of Iain Macintyre, FRS. Bone. 2002, 30: 655–663.
Zaidi M, Chambers TJ, Bevis PJ, et a1. Effects of peptides from the ealcitonin genes on bone and bone cells. J Exp Physiol. 1988.73: 471-485.
陈孝文,江黎明,叶锋.肾内分泌学.广州:广东科技出版社.1994,81-98.
陈云松张学成孙平楠.转鲑鱼降钙素基因酵母的亚急性毒性实验.中国海洋大学学报. 2007, 37(6 Sup.):109-114
郭世绂,罗先正,邱贵兴.骨质疏松—基础与临床[M].天津:天津科学技术出版社.2001, 443-447.
郭世绂.骨转换的力学因素与骨质疏松(一)骨转换.中国骨肿瘤骨病.2002 ,1 (1):61-64.
郭世绂.骨转换的力学因素与骨质疏松(二)骨组织与力学环境.中国骨肿瘤骨病. 2002,1(2):119-123.
刘江涛,王永清,张卫国等.用酶消化-连续组织块法培养SD大鼠成骨细胞的研究.中国中医骨伤科杂志. 2006,14(6):46-48
司徒镇强,吴军正.细胞培养.西安:世界图书出版西安公司. 2004,83-84.
孙安阳,俞彰,钟慈声等.大鼠动脉钙负荷模型的建立及确证.中华医学杂志. 1999,79(10):769-772.
孙平楠周小玲张学成. 1, 25-二羟维生素D3法建立高钙血症大鼠模型的研究..现代生物医学进展. 2009, 9(3):65-67
王玉梅,张学成,周一江,等. cpcB与CT融合蛋白表达质粒在大肠杆菌中的表达和纯化.高技术通讯. 2005, 15: 63-66.
彦世铭,洪昭毅,李增禧.实用元素医学.郑州:河南医科大学出版社.1999,90-99.
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