1、核因子—κB受体激活物配基（RANKL）反义RNA影响破骨细胞样细胞生成的研究 2、小鼠RANKL活性区cDNA克隆及在大肠杆菌和毕赤酵母中的表达 3、人护骨素（OPG）成熟肽基因酵母表达载体的构建
|
摘要
护骨素(osteoprotegerin，OPG)及其同源配基核因子-κB受体激活物配基(receptor activator of nuclear factor κB ligand，RANKL)在破骨细胞分化和激活的调控中具有十分关键的作用。它们都由成骨细胞／骨髓基质细胞谱系表达。在允许浓度的M-CSF存在时，RANKL有力促进破骨细胞的分化、成熟和激活，并抑制细胞凋亡，是这些过程所必需的终极效应分子。OPG是RANKL的诱骗受体，通过与表达于成骨细胞／骨髓基质谱系表面的RANKL结合阻断RANKL对破骨细胞生成等的诱导作用。多数趋钙激素和细胞因子通过影响RANKL与OPG的相对表达水平来调控破骨细胞的分化和功能。
本实验通过研究RANKL和OPGmRNA在OVX小鼠骨组织中的相对表达水平，探讨RANKL和OPG在PMO发病中的意义。
我们将40只6周龄昆明小鼠随机分为5组，分别为Ⅰ组：对动物行假手术，术后1周处死；Ⅱ组：行卵巢切除手术，安慰剂治疗，术后1周处死；Ⅲ组：行卵巢切除手术，安慰剂治疗，术后5周处死；Ⅳ组：行卵巢切除手术，术后给苯甲酸雌二醇治疗，5周后处死；Ⅴ组：行假手术，术后5周处死。动物处死后，提取胫、股骨骨组织总RNA，以GAPDH作为内参照，RT-PCR扩增检测OVX小鼠RANKL和OPG mRNA表达水平，发现手术后第1、5周去卵巢小鼠骨组织RANKL mRNA表达水平都较假手术组明显增高，采用雌激素治疗可显著降低RANKL mRNA表达水平。而OPG mRNA表达水平在去卵巢后第1周时较假手术组显著降低，在第5周时却比假手术组显著升高，可能是机体的一种代偿机制。雌激素治疗组则介于去卵巢5周组和假手术组之间，但与二者均无显著性差异。以RANKL／OPG来表示RANKL和OPG mRNA的相对表达水平，我们发现，去卵巢后，RANKL／OPG显著增高，采用雌激素防
天津医科大学博士学位论文
治可使RANKL／OPG与假手术组无显著差异。
本研究结果提示PMO发病中雌激素减退所导致的骨丢失与骨组织RANKL
和OPG的mRNA相对表达水平紊乱有关。
Receptor Activator of Nuclear Factor-Kappa B Ligand (RANKL), a membrane-bound protein expressed on osteoblasts and bone marrow stromal cells,is believed to induce osteoclast differentiation and activation by direct cell to cell interaction with osteoclast lineage cells.This induction is interrupted by osteoprotegerin (OPG),a soluble receptor of RANKL. Thus, OPG conteracts the stimulatory effect of RANKL on osteoclast differentiation and activation and it has been suggested that a high RANKL/OPG ratio is associated with an increased osteoclatogenesis while a low ratio with a decreased osteoclatogenesis. Previous in vitro studies indiated that other osteotropic hormones and factors such as PTH, 1, 25(OH)2D3, IL-1, IL-6 and TNF regulated osteoclastogenesis through the effect on RANKL/OPG ratio. The aim of the present study was to investigate the expression and regulation of RANKL and OPG in vivo in ovariectomized mice.
forty female mice at six weeks of age were allocated into 5 groups, 8 in each. Group II > El and IVwere ovariectomized. Group IV were injected subcutaneusly with Benzoic estradiol for 5 weeks
taneusly with Benzoic estradiol for 5 weeks
one day after the ovariectomy. Group II and III were given placebo instead and sacrificed 1 and 5 weeks after the ovariectomy respectively. Group I and V were sham-operated and treated with placebo,then sacrificed l,and 5 weeks after ovariectomy. The tibia and femor were dissected out and mRNA levels of RANKL and OPG were studied using reverse transcription polyraerase chain reaction technique. The data showed that the mRNA level of RANKL was increased by ovariectomy while that of OPG was reduced in one week but increased in 5 weeks after ovariectomy, which seemed a compensation to the acceleration of bone loss. As a consequence, the ratio RANKL/OPG was increased by ovariectomy while reduced significantly when Benzoic estradiol was administrated.
In summary, Our results indicate that Ovx-induced bone loss may be associated with the disturbance of RANKL/OPG.
本实验通过研究RANKL和OPGmRNA在OVX小鼠骨组织中的相对表达水平，探讨RANKL和OPG在PMO发病中的意义。
我们将40只6周龄昆明小鼠随机分为5组，分别为Ⅰ组：对动物行假手术，术后1周处死；Ⅱ组：行卵巢切除手术，安慰剂治疗，术后1周处死；Ⅲ组：行卵巢切除手术，安慰剂治疗，术后5周处死；Ⅳ组：行卵巢切除手术，术后给苯甲酸雌二醇治疗，5周后处死；Ⅴ组：行假手术，术后5周处死。动物处死后，提取胫、股骨骨组织总RNA，以GAPDH作为内参照，RT-PCR扩增检测OVX小鼠RANKL和OPG mRNA表达水平，发现手术后第1、5周去卵巢小鼠骨组织RANKL mRNA表达水平都较假手术组明显增高，采用雌激素治疗可显著降低RANKL mRNA表达水平。而OPG mRNA表达水平在去卵巢后第1周时较假手术组显著降低，在第5周时却比假手术组显著升高，可能是机体的一种代偿机制。雌激素治疗组则介于去卵巢5周组和假手术组之间，但与二者均无显著性差异。以RANKL／OPG来表示RANKL和OPG mRNA的相对表达水平，我们发现，去卵巢后，RANKL／OPG显著增高，采用雌激素防
天津医科大学博士学位论文
治可使RANKL／OPG与假手术组无显著差异。
本研究结果提示PMO发病中雌激素减退所导致的骨丢失与骨组织RANKL
和OPG的mRNA相对表达水平紊乱有关。
Receptor Activator of Nuclear Factor-Kappa B Ligand (RANKL), a membrane-bound protein expressed on osteoblasts and bone marrow stromal cells,is believed to induce osteoclast differentiation and activation by direct cell to cell interaction with osteoclast lineage cells.This induction is interrupted by osteoprotegerin (OPG),a soluble receptor of RANKL. Thus, OPG conteracts the stimulatory effect of RANKL on osteoclast differentiation and activation and it has been suggested that a high RANKL/OPG ratio is associated with an increased osteoclatogenesis while a low ratio with a decreased osteoclatogenesis. Previous in vitro studies indiated that other osteotropic hormones and factors such as PTH, 1, 25(OH)2D3, IL-1, IL-6 and TNF regulated osteoclastogenesis through the effect on RANKL/OPG ratio. The aim of the present study was to investigate the expression and regulation of RANKL and OPG in vivo in ovariectomized mice.
forty female mice at six weeks of age were allocated into 5 groups, 8 in each. Group II > El and IVwere ovariectomized. Group IV were injected subcutaneusly with Benzoic estradiol for 5 weeks
taneusly with Benzoic estradiol for 5 weeks
one day after the ovariectomy. Group II and III were given placebo instead and sacrificed 1 and 5 weeks after the ovariectomy respectively. Group I and V were sham-operated and treated with placebo,then sacrificed l,and 5 weeks after ovariectomy. The tibia and femor were dissected out and mRNA levels of RANKL and OPG were studied using reverse transcription polyraerase chain reaction technique. The data showed that the mRNA level of RANKL was increased by ovariectomy while that of OPG was reduced in one week but increased in 5 weeks after ovariectomy, which seemed a compensation to the acceleration of bone loss. As a consequence, the ratio RANKL/OPG was increased by ovariectomy while reduced significantly when Benzoic estradiol was administrated.
In summary, Our results indicate that Ovx-induced bone loss may be associated with the disturbance of RANKL/OPG.
引文
1． Tsuda E, Goto M, Monochizuki S-I, et al. Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis. Biochemical and Biophysical Research Communication.1997;234:137-142
2． Simonet WS, Lacey DL, Dunstan CR, et al._Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 1997 Apr 18;89(2) :309-19
3． Akatsu T, Murakami T, Nishikawa M, et al.Osteoclastogenesis-inhibitory factor suppresses osteoclast survival by interfering in the interaction of stromal cells with osteoclast. Biochemical and Biophysical Research Communcation. 1998:250:229-234
4． Tsukii K, Shima N, Mochizuki S, et al. Osteoclast differentiation factor mediates an essential signal for bone resorption induced by 1 alpha, 25-dihydroxyvitamin D3, prostaglandin E2, or parathyroid hormone in the microenvironment of bone. Biochem Biophys Res Commun 1998 May 19;246(2) :337-41
5． Hakeda Y, Kobayashi Y, Yamaguchi K, et al. Osteoclastogenesis-inhibitory factor directly inhibits bone-resorbing activity of isolated mature osteoclasts. Biochem Biophys Res Commun. 1998:251:796-801
6． Yasuda H, Shima N, Nakagawa N, et al. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 1998 Mar;139(3) :1329-37
7． Bucay N, Sarosi I, Dunstan CR, et al. osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 1998 May 1; 12 (9) :1260-8
8． Lacey DL, Timms E, Tan HL,et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998 Apr 17;93(2) :165-76
9． Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 1998 Mar 31:95(7) :3597-602
10． Hofbauer LC, Khosla S, Dunstan CR, et al. Estrogen stimulate gene expression and protein production of osteoprotegerin in human osteoblastic cells. Endocrinology 1999:140:4367-4370
11． Quinn JM, Elliott J, Gillespie MT, et al. A combination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro. Endocrinology 1998 Oct;139(10) :4424-7
12． Fuller K, Wong B, Fox S,et al. TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J Exp Med 1998 Sep 7;188(5) :997-1001
13． Burgess TL, Qian Y, Kaufman S, et al. The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J Cell Biol 1999 May 3; 145(3) :527-38
14． Kong YY, Feige U, Sarosi I, et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 1999 Nov 18:402(6759) :304-9
15． Yano K, Tsuda E, Washida, N, et al.Immunological characterization of circulating osteoprotegerin/osteoclastogenesis inhibitory factor:increase serum concentration in postmenopausal women with osteoporosis.J Bone Miner Res;14:518-527
16． Hofbauer LC, Lacey DL, Dunstan CR, et, al. Interleukin-1beta and tumor necrosis factor-alpha, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells. Bone 1999 Sep;25(3) :255-9
17． Lorenz C. Hofbauer, Francescar Gori,B. Lawrence Riggs,et alStimulation of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells:potential paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinology. 1999:140(10) :4382-4389
18． Murakami T, Yamamoto M, Ono K, et al. Transforming growth factor-betal increases mRNA levels of osteoclastogenesis inhibitory factor in osteoblastic/stromal cells and inhibits the survival of murine osteoclast-like cells. Biochem Biophys Res Commun 1998 Nov 27;252 (3) :747-52
19．李卫东，黄凤岐，谭郁彬，等．正常和去势大鼠骨组织内雌激素受体mRNA表达水平的研究．中华病理学杂志．1995；24(5)；312-314
20．郭若霖，郭善一，鲍秋野，等(Guo R L,Guo S Y,Bao Q Y et al.)。竞争性RTPCR测定法：BMP-2 mRNA的定量检测。中国生物化学与分子生物学报 2000，16(5) ：680-683
21. Hsu H, Lacey DL, Dunstan CR, et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3540-5
22. Nemeth GG, Heydemann A, Bolander ME. Isolation and Analysis of Ribonucleic Acids from Skeletal Tissues. Anal Biochem 1989;183,301-304
23. Ikeda T, Yamaguchi A, Yokose S, et al. Change in biological activity of bone cells in ovariectomized rats revealed by in situ hibridization. J Bone Miner Res. 11:780-788
24. Mieko Saika, Daisuke Inoue, Shinsuke Kido, et al. 17β-estradiol stimulates expression of osteoprotegerin by a mouse stromal cell line, ST2, via estragen receptor-α .Endocrinology. 2001;142(5):2205-2212
25. Kitazawa R, Kitazawa, S, Maeda, S. Biochim Biophysi Acta. 1999;1445:134-141
26. Srivastava S, Toraldo G, Weitzmann MN, et al. Estragen decrease osteoclast formation by down regulating receptor activator of NF-к B ligand(RANKL)-induced JNK activation. J Biol Chem. 2001:276(12):8836-8840
27. Cenci S, Weitzmann MN, Roggia C, et ,al. Estrogen deficiency induces bone loss by enhancing T cell production of TNF-α .J clin Invest. 2000; 106(10):1229-1237
28. Roggia C, Gao Y, Cenci S, et al. Up-regulation of TNF-producing T cells in the bone marrow: a key mechanism by which estrogen deficiency induces bone loss in vivo. PNAS. 2001;98(24):13960-13965
29. Hofbauer LC, Gori F, Riggs BL, et al. Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic cell lineage cells:potential paracrine mechnisms of glucocorticoids induced osteoporosis. Endocrinology. 1999;140:4382-4389
30. Sasaki N, Kusano E, Ando Y, et al. Glucocorticoid decreases circulating osteoprotegerin(OPG):possible mechnism for. glucocorticoids induced
osteoporosis. Nephrol Dial Transplant. 2001;16:479-82
31． Takayanagi H,Iizuka H,Juji T, et al. Involvement of recepror activator of nuclear factor k B ligand/osteoclate differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis. Arthritis Rheum. 2000:43:259-69
32． Horwood NJ, Kartsogiannis V Quinn JMW,et al. Activated T cells support osteoclast formation in vitro. Biochem Biophys Res Commun. 1999:265:144-150
33． Gravallese EM, Manning C, Tsay A, et al. Involvement of recepror activator of nuclear factor K B ligand/osteoclate differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis. Arthritis Rheum. 2000:43:250-258
34． Lee SK, Lorenzo JA,Parthyrioid hormone stimulates TRANCE and inhibits osteoprotegerin messenger ribonucleic acid expression in murine bone marrow cultures correlation osteoclast-like cell formation. Endocrionology. 1999:140:3552-61
35． Horwood NJ, Elliott J,Martin TJ,et al. Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells. Endocrinology. 1998:139:4743-6
36． Henna C, Barsony J, Reddy SV, et al. 1,25-Dihydroxyvitamin D3 hypersensitivity of osteoclast precursors from patient with Paget's disease.J Bone Miner Res. 2000;15:228-236
37． Menna C, Reddy SV, Kurihara N, et al.Enhanced RANK ligand expression and responsibility of bone marrow cells in Paget's disease of bone.J Clin Invest. 2000;105:1833-8．
38． 罗超权.基因诊断与基因治疗进展.河南医科大学出版社,2000年2月第一版。
39． Gelband CH, Reaves PY, Evans J, et al. Angiotensin II type 1 receptor antisense gene therapy prevents altered renal vascular calcium homeostasis in hypertension. Hypertension. 1999:33:360-365
40． Raizada MK, Katovich MJ, Wang HW, et al. Is antisense gene therapy a step in the right direction in the control of hypertension. AJP. 1999:277(2) :423-432
41． Laitala T, Vaananen K. Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or
two subunits of vacuolar H-ATPase. J Clin Invest. 1994;93:2311-2318
42. Takayanagi H, Juji T, Miyazaki T, et al. Suppression of arthritic bone destruction by adenovirus-mediated csk gene gene transfer5 to synoviocytes and osteoclasts. J Clin Invest. 1999;94(2):137-145
43. Robert L. Jilka, Giao Hangoc, Giuseppe Girasole, et al. Increased osteoclast development after estrogen loss:mediation by interleukin6. Science. 1992:257:88-91
44. Girasole G, Jilka RL, Passeri G, et al. 17 beta-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens. J Clin Invest. 1992;89(3):883-91
45. Poli V, Balena R, Fattori E, et al. Interleukin-6 deficient mice are protected from bone loss caused by estrogen depletion. EMBO-J. 1994;13(5):1189-96
46. Ammann-P, Rizzoli R, Bonjour JP, et al. Transgenic mice expressing soluble tumor necrosis factor-receptor are protected against bone loss caused by estrogen deficiency. J Clin Invest. 1997;99(7): 1699-703
47. Kimble RB, Bain S, Pacifici R, et al. The functional block of TNF but not of IL-6 prevents bone loss in ovariectomized mice. J Bone Miner Res. 1997;12(6):935-41
48. Bismar H, Diel I, Ziegler R, et al. Increased cytokine secretion by human bone marrow cells after menopause or discontinuation of estrogen replacement. J Clin Endocrinol Metab. 1995;80(11):3351-5
49．金冬雁，黎孟枫等译。分子克隆实验指南。1992年12月。科学出版社。
50．姜泊，张亚历主编．分子生物学常用实验方法．1996年2月．第一版人民军医出版社
51．B．D．哈密斯等 蛋白质凝胶电泳实践方法 第一版科学出版社 1986
52．王重庆，李云兰，李德昌主编．高级生物化学实验教程．第一版 1994 北京大学出版社
53. Towbin H, Staehelin T and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:Procedure and some applications. Proc Natl Acad Sci 1979;76:4350
54. J.P. Dean Goldring, Lillian Ravaioli, Solubilization of Protein-Dye complexes on nitrocellulose to quantity proteins spectrophotometrically. Analytical biochemistry 242,197-201(1996)
55. Smith DB, Johnson KS , Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988;67(1):31-40
56. Smith PK. Krohn RI. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76
57．孙树汉主编．基因工程原理与方法．2001年7月．人民卫生出版社。
58．卢圣栋主编。现代分子生物学实验技术第二版。1999年12月。中国协和医科大学出版社。
59．颜子颖、王海林译。精编分子生物学实验指南。1998年6月。科学出版社。
60. Clare JJ, Rayment FB, Ballantine S PA, et al. High-level Expression of Tetanus Toxin Fragment c in Pichia pastoris Strains Containing Multiple Tandem Integrations of the Gene. Bio/Technology. 1991; 9:455-460
60. Couderc R, Baratti J. Oxidation of methanol by the yeast pichia pastoris:purification and properties of alcohol oxidase. Agric Biol Chem. 1980,44:2279-2289
61. Grinna LS, and Tschopp JF. Size Distribution and General Structural Features of N-Linked Oligosaccharides from the Methylotrophic Yeast, Pichia pastoris. Yeast .1989;5:107-115
62. Tschopp JF, Sverlow G, Kosson R, et al. High Level Secretion of Glycosylated Invertase in the Methylotrophic Yeast Pichia pastoris. Bio/Technology. 1987; 5:1305-1308
63. Ellis SB, Brust PF, Koutz PJ, et al. Isolation of Alcohol Oxidase and Two other Methanol Regulatable Genes from the Yeast, Pichia pastoris. Mol. Cell. Biol.1985;5: 1111-1121.
64. Koutz PJ, Davis GR, Stillman C, et al. Structural Comparison of the Pichia pastoris Alcohol Oxidase Genes. Yeast. 1989;5:167-177
65．田昱，李平，朱运松．人纤溶酶原激活剂抑制物2型(PAI-2)在Pichia pastoris中的表达．中国生物化学与分子生物学报．1998；14(3)：259-263
66．邓兵兵，方宏清，薛冲，等．人肝细胞生长因子(hdHGF)基因在毕赤酵母中的分泌表达．中国生物化学与分子生物学报．2001；17(5)：590-594
67. Sreekrishna K., Nelles L, Potenz R, et al. High Level Expression, Purification, and Characterization of Recombinant Human Tumor Necrosis Factor Synthesized in the Methylotrophic Yeast Pichia pastoris.
Biochemistry .1989:28: 4117-4125
68． Weiss HM, Haase W, Michel H, et al. Expression of Functional Mouse 5-HT 5A Serotonin Receptor in the Methylotrophic Yeast Pichia pastoris: Pharmacological Characterization and Localization. FEBS. 1995; 377: 451-456．
69． Lopes TS,Klootwijk J, Veenstra AE, et al. High-copy-number integration into the ribosomal DNA of Sacchromyces cerevisiae.Gene. 1989:79(2) :199-206
70． Lopes TS, Hakkaart G-TAJ, Koerts BL, et al. Mechnisms of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Sacchromyces cerevisiae. Gene. 1991;105(1) :83-90
71． 唐南筠，霍克克，李育阳.乳酸克鲁维酵母高拷贝整合载体的构建和应用.生物化学与生物物理学报, 1996:28 (5) : 540-545
72． Kondo K, Miura Y, Sone H,et al. High level expression of a sweet protein, Monellin, in the food yeast Candida Utilis. Nature Biotechnology. 1997;15(5) :453-457
73． Huang L, Xu J, Wood DJ, et al.Gene expression of osteoprotegerin ligand ,osteoprotegerin and receptor activator of NF-K B in giant cell tumor of bone:possible involvement in tumor cell-induced osteoclast like cell formation. Am J Pathol. 2000;156:761-767
74． Atkins GJ,Haynes DR, Graves SE, et al. Expression of osteoclast differentiation signals by stromal elements of giant cell tumors. J Bonr Miner Res.2000;15:640-649
75． O'Brien CA, Gubrij I, Lin SC, et al. STAT3 activation in stromal/osteoblastic. cells is required for induction of the receptor activator of NF-K B ligand and stimulation of osteoclastogenesis by gp130-utilizing cytokines or interleukin-1 but not 1,25-dihydroxyvitaminD3 or parathyroid hormone. J Biol Chem. 1999; 274:19301-19308
76． Greenfield EM. ODF/OPGL expression is regulated by IL-1 and IL-6． J Bone Miner Res.1999;14(suppl 1) :SA211
77． Thomas RJ, Guise TA, Yin JJ,et al. Breast cancer cells interact with osteoblasts to support osteoclast formation. Endocrinology.1999;140:4451-4458
78． Kitazawa S, Mori H, Maeda S, et al. Osteoclast differentiation factor (ODF/OPGL/RANKL/TRANCE) expression in bone invasion model of breast cancer. J Bone Miner Res. 1999; 14(suppl 1) :1083
79． Westin JS, Ljunghall S, Nilsson K, et al. Human multiple myeoloma cell lines express mRNA for osteoclast differentiation factor. J Bone Miner Res. 1999:14(suppl 1) :F063
80． Chikatsu N, Takeuchi Y, Tamura Y, et al. Interaction between cancer and bone marrow cells induce osteoclast differentiation factor expression and osteoclast like cell formation in vitro. Biochem Biophys Res Commun. 2000 ; 267:632-637
81． Nagai M, Kyakumato S, Sato N. Cancer cell responsible for humoral hypercalcemia express mRNA encoding a secreted form of ODF/TRANCE that induces osteoclast formation. Biochem Biophys Res Commun. 2000 ; 269 : 532-536
82． Capparelli C, Kostenuik PJ, Morony S, et al. Osteoprotegerin prevents and reverses hypercalcemia in a murine model of humormal hypercalcemia of malignancy. Cancer Res. 2000 ; 60 : 783-787
83． Morony S, Capparelli C, Kostenuik PJ, et al. Osteoprotegerin prevents osteolytic bone destruction in both athymic and synergeneic models of experimental tumor metastasis to bone. J Bone Miner Res. 1999; 14(suppl 1) :1125
84． Honore P, Luger NM, Sabino MAC, et al. Osteoprotegerin blocks bone cancer induced skeletal destruction, skeletal pain and pain-ralated neurochemical reorganization of the spinal cord. Nat Med. 2000;5:521-528
85． Bucay N, Sarosi I.Dunstan CR, et al. Osteoprotegerin deficent mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998; 12:1260-1268
86． Min H, Morony S, Sarosi I, et al. Osteoprotegerin reverses osteoporosis by inhibiting endpsteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis. J Exp Med. 2000; 192:463-474
2． Simonet WS, Lacey DL, Dunstan CR, et al._Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 1997 Apr 18;89(2) :309-19
3． Akatsu T, Murakami T, Nishikawa M, et al.Osteoclastogenesis-inhibitory factor suppresses osteoclast survival by interfering in the interaction of stromal cells with osteoclast. Biochemical and Biophysical Research Communcation. 1998:250:229-234
4． Tsukii K, Shima N, Mochizuki S, et al. Osteoclast differentiation factor mediates an essential signal for bone resorption induced by 1 alpha, 25-dihydroxyvitamin D3, prostaglandin E2, or parathyroid hormone in the microenvironment of bone. Biochem Biophys Res Commun 1998 May 19;246(2) :337-41
5． Hakeda Y, Kobayashi Y, Yamaguchi K, et al. Osteoclastogenesis-inhibitory factor directly inhibits bone-resorbing activity of isolated mature osteoclasts. Biochem Biophys Res Commun. 1998:251:796-801
6． Yasuda H, Shima N, Nakagawa N, et al. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 1998 Mar;139(3) :1329-37
7． Bucay N, Sarosi I, Dunstan CR, et al. osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 1998 May 1; 12 (9) :1260-8
8． Lacey DL, Timms E, Tan HL,et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998 Apr 17;93(2) :165-76
9． Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 1998 Mar 31:95(7) :3597-602
10． Hofbauer LC, Khosla S, Dunstan CR, et al. Estrogen stimulate gene expression and protein production of osteoprotegerin in human osteoblastic cells. Endocrinology 1999:140:4367-4370
11． Quinn JM, Elliott J, Gillespie MT, et al. A combination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro. Endocrinology 1998 Oct;139(10) :4424-7
12． Fuller K, Wong B, Fox S,et al. TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J Exp Med 1998 Sep 7;188(5) :997-1001
13． Burgess TL, Qian Y, Kaufman S, et al. The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J Cell Biol 1999 May 3; 145(3) :527-38
14． Kong YY, Feige U, Sarosi I, et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 1999 Nov 18:402(6759) :304-9
15． Yano K, Tsuda E, Washida, N, et al.Immunological characterization of circulating osteoprotegerin/osteoclastogenesis inhibitory factor:increase serum concentration in postmenopausal women with osteoporosis.J Bone Miner Res;14:518-527
16． Hofbauer LC, Lacey DL, Dunstan CR, et, al. Interleukin-1beta and tumor necrosis factor-alpha, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells. Bone 1999 Sep;25(3) :255-9
17． Lorenz C. Hofbauer, Francescar Gori,B. Lawrence Riggs,et alStimulation of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells:potential paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinology. 1999:140(10) :4382-4389
18． Murakami T, Yamamoto M, Ono K, et al. Transforming growth factor-betal increases mRNA levels of osteoclastogenesis inhibitory factor in osteoblastic/stromal cells and inhibits the survival of murine osteoclast-like cells. Biochem Biophys Res Commun 1998 Nov 27;252 (3) :747-52
19．李卫东，黄凤岐，谭郁彬，等．正常和去势大鼠骨组织内雌激素受体mRNA表达水平的研究．中华病理学杂志．1995；24(5)；312-314
20．郭若霖，郭善一，鲍秋野，等(Guo R L,Guo S Y,Bao Q Y et al.)。竞争性RTPCR测定法：BMP-2 mRNA的定量检测。中国生物化学与分子生物学报 2000，16(5) ：680-683
21. Hsu H, Lacey DL, Dunstan CR, et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3540-5
22. Nemeth GG, Heydemann A, Bolander ME. Isolation and Analysis of Ribonucleic Acids from Skeletal Tissues. Anal Biochem 1989;183,301-304
23. Ikeda T, Yamaguchi A, Yokose S, et al. Change in biological activity of bone cells in ovariectomized rats revealed by in situ hibridization. J Bone Miner Res. 11:780-788
24. Mieko Saika, Daisuke Inoue, Shinsuke Kido, et al. 17β-estradiol stimulates expression of osteoprotegerin by a mouse stromal cell line, ST2, via estragen receptor-α .Endocrinology. 2001;142(5):2205-2212
25. Kitazawa R, Kitazawa, S, Maeda, S. Biochim Biophysi Acta. 1999;1445:134-141
26. Srivastava S, Toraldo G, Weitzmann MN, et al. Estragen decrease osteoclast formation by down regulating receptor activator of NF-к B ligand(RANKL)-induced JNK activation. J Biol Chem. 2001:276(12):8836-8840
27. Cenci S, Weitzmann MN, Roggia C, et ,al. Estrogen deficiency induces bone loss by enhancing T cell production of TNF-α .J clin Invest. 2000; 106(10):1229-1237
28. Roggia C, Gao Y, Cenci S, et al. Up-regulation of TNF-producing T cells in the bone marrow: a key mechanism by which estrogen deficiency induces bone loss in vivo. PNAS. 2001;98(24):13960-13965
29. Hofbauer LC, Gori F, Riggs BL, et al. Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic cell lineage cells:potential paracrine mechnisms of glucocorticoids induced osteoporosis. Endocrinology. 1999;140:4382-4389
30. Sasaki N, Kusano E, Ando Y, et al. Glucocorticoid decreases circulating osteoprotegerin(OPG):possible mechnism for. glucocorticoids induced
osteoporosis. Nephrol Dial Transplant. 2001;16:479-82
31． Takayanagi H,Iizuka H,Juji T, et al. Involvement of recepror activator of nuclear factor k B ligand/osteoclate differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis. Arthritis Rheum. 2000:43:259-69
32． Horwood NJ, Kartsogiannis V Quinn JMW,et al. Activated T cells support osteoclast formation in vitro. Biochem Biophys Res Commun. 1999:265:144-150
33． Gravallese EM, Manning C, Tsay A, et al. Involvement of recepror activator of nuclear factor K B ligand/osteoclate differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis. Arthritis Rheum. 2000:43:250-258
34． Lee SK, Lorenzo JA,Parthyrioid hormone stimulates TRANCE and inhibits osteoprotegerin messenger ribonucleic acid expression in murine bone marrow cultures correlation osteoclast-like cell formation. Endocrionology. 1999:140:3552-61
35． Horwood NJ, Elliott J,Martin TJ,et al. Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells. Endocrinology. 1998:139:4743-6
36． Henna C, Barsony J, Reddy SV, et al. 1,25-Dihydroxyvitamin D3 hypersensitivity of osteoclast precursors from patient with Paget's disease.J Bone Miner Res. 2000;15:228-236
37． Menna C, Reddy SV, Kurihara N, et al.Enhanced RANK ligand expression and responsibility of bone marrow cells in Paget's disease of bone.J Clin Invest. 2000;105:1833-8．
38． 罗超权.基因诊断与基因治疗进展.河南医科大学出版社,2000年2月第一版。
39． Gelband CH, Reaves PY, Evans J, et al. Angiotensin II type 1 receptor antisense gene therapy prevents altered renal vascular calcium homeostasis in hypertension. Hypertension. 1999:33:360-365
40． Raizada MK, Katovich MJ, Wang HW, et al. Is antisense gene therapy a step in the right direction in the control of hypertension. AJP. 1999:277(2) :423-432
41． Laitala T, Vaananen K. Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or
two subunits of vacuolar H-ATPase. J Clin Invest. 1994;93:2311-2318
42. Takayanagi H, Juji T, Miyazaki T, et al. Suppression of arthritic bone destruction by adenovirus-mediated csk gene gene transfer5 to synoviocytes and osteoclasts. J Clin Invest. 1999;94(2):137-145
43. Robert L. Jilka, Giao Hangoc, Giuseppe Girasole, et al. Increased osteoclast development after estrogen loss:mediation by interleukin6. Science. 1992:257:88-91
44. Girasole G, Jilka RL, Passeri G, et al. 17 beta-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens. J Clin Invest. 1992;89(3):883-91
45. Poli V, Balena R, Fattori E, et al. Interleukin-6 deficient mice are protected from bone loss caused by estrogen depletion. EMBO-J. 1994;13(5):1189-96
46. Ammann-P, Rizzoli R, Bonjour JP, et al. Transgenic mice expressing soluble tumor necrosis factor-receptor are protected against bone loss caused by estrogen deficiency. J Clin Invest. 1997;99(7): 1699-703
47. Kimble RB, Bain S, Pacifici R, et al. The functional block of TNF but not of IL-6 prevents bone loss in ovariectomized mice. J Bone Miner Res. 1997;12(6):935-41
48. Bismar H, Diel I, Ziegler R, et al. Increased cytokine secretion by human bone marrow cells after menopause or discontinuation of estrogen replacement. J Clin Endocrinol Metab. 1995;80(11):3351-5
49．金冬雁，黎孟枫等译。分子克隆实验指南。1992年12月。科学出版社。
50．姜泊，张亚历主编．分子生物学常用实验方法．1996年2月．第一版人民军医出版社
51．B．D．哈密斯等 蛋白质凝胶电泳实践方法 第一版科学出版社 1986
52．王重庆，李云兰，李德昌主编．高级生物化学实验教程．第一版 1994 北京大学出版社
53. Towbin H, Staehelin T and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:Procedure and some applications. Proc Natl Acad Sci 1979;76:4350
54. J.P. Dean Goldring, Lillian Ravaioli, Solubilization of Protein-Dye complexes on nitrocellulose to quantity proteins spectrophotometrically. Analytical biochemistry 242,197-201(1996)
55. Smith DB, Johnson KS , Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988;67(1):31-40
56. Smith PK. Krohn RI. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76
57．孙树汉主编．基因工程原理与方法．2001年7月．人民卫生出版社。
58．卢圣栋主编。现代分子生物学实验技术第二版。1999年12月。中国协和医科大学出版社。
59．颜子颖、王海林译。精编分子生物学实验指南。1998年6月。科学出版社。
60. Clare JJ, Rayment FB, Ballantine S PA, et al. High-level Expression of Tetanus Toxin Fragment c in Pichia pastoris Strains Containing Multiple Tandem Integrations of the Gene. Bio/Technology. 1991; 9:455-460
60. Couderc R, Baratti J. Oxidation of methanol by the yeast pichia pastoris:purification and properties of alcohol oxidase. Agric Biol Chem. 1980,44:2279-2289
61. Grinna LS, and Tschopp JF. Size Distribution and General Structural Features of N-Linked Oligosaccharides from the Methylotrophic Yeast, Pichia pastoris. Yeast .1989;5:107-115
62. Tschopp JF, Sverlow G, Kosson R, et al. High Level Secretion of Glycosylated Invertase in the Methylotrophic Yeast Pichia pastoris. Bio/Technology. 1987; 5:1305-1308
63. Ellis SB, Brust PF, Koutz PJ, et al. Isolation of Alcohol Oxidase and Two other Methanol Regulatable Genes from the Yeast, Pichia pastoris. Mol. Cell. Biol.1985;5: 1111-1121.
64. Koutz PJ, Davis GR, Stillman C, et al. Structural Comparison of the Pichia pastoris Alcohol Oxidase Genes. Yeast. 1989;5:167-177
65．田昱，李平，朱运松．人纤溶酶原激活剂抑制物2型(PAI-2)在Pichia pastoris中的表达．中国生物化学与分子生物学报．1998；14(3)：259-263
66．邓兵兵，方宏清，薛冲，等．人肝细胞生长因子(hdHGF)基因在毕赤酵母中的分泌表达．中国生物化学与分子生物学报．2001；17(5)：590-594
67. Sreekrishna K., Nelles L, Potenz R, et al. High Level Expression, Purification, and Characterization of Recombinant Human Tumor Necrosis Factor Synthesized in the Methylotrophic Yeast Pichia pastoris.
Biochemistry .1989:28: 4117-4125
68． Weiss HM, Haase W, Michel H, et al. Expression of Functional Mouse 5-HT 5A Serotonin Receptor in the Methylotrophic Yeast Pichia pastoris: Pharmacological Characterization and Localization. FEBS. 1995; 377: 451-456．
69． Lopes TS,Klootwijk J, Veenstra AE, et al. High-copy-number integration into the ribosomal DNA of Sacchromyces cerevisiae.Gene. 1989:79(2) :199-206
70． Lopes TS, Hakkaart G-TAJ, Koerts BL, et al. Mechnisms of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Sacchromyces cerevisiae. Gene. 1991;105(1) :83-90
71． 唐南筠，霍克克，李育阳.乳酸克鲁维酵母高拷贝整合载体的构建和应用.生物化学与生物物理学报, 1996:28 (5) : 540-545
72． Kondo K, Miura Y, Sone H,et al. High level expression of a sweet protein, Monellin, in the food yeast Candida Utilis. Nature Biotechnology. 1997;15(5) :453-457
73． Huang L, Xu J, Wood DJ, et al.Gene expression of osteoprotegerin ligand ,osteoprotegerin and receptor activator of NF-K B in giant cell tumor of bone:possible involvement in tumor cell-induced osteoclast like cell formation. Am J Pathol. 2000;156:761-767
74． Atkins GJ,Haynes DR, Graves SE, et al. Expression of osteoclast differentiation signals by stromal elements of giant cell tumors. J Bonr Miner Res.2000;15:640-649
75． O'Brien CA, Gubrij I, Lin SC, et al. STAT3 activation in stromal/osteoblastic. cells is required for induction of the receptor activator of NF-K B ligand and stimulation of osteoclastogenesis by gp130-utilizing cytokines or interleukin-1 but not 1,25-dihydroxyvitaminD3 or parathyroid hormone. J Biol Chem. 1999; 274:19301-19308
76． Greenfield EM. ODF/OPGL expression is regulated by IL-1 and IL-6． J Bone Miner Res.1999;14(suppl 1) :SA211
77． Thomas RJ, Guise TA, Yin JJ,et al. Breast cancer cells interact with osteoblasts to support osteoclast formation. Endocrinology.1999;140:4451-4458
78． Kitazawa S, Mori H, Maeda S, et al. Osteoclast differentiation factor (ODF/OPGL/RANKL/TRANCE) expression in bone invasion model of breast cancer. J Bone Miner Res. 1999; 14(suppl 1) :1083
79． Westin JS, Ljunghall S, Nilsson K, et al. Human multiple myeoloma cell lines express mRNA for osteoclast differentiation factor. J Bone Miner Res. 1999:14(suppl 1) :F063
80． Chikatsu N, Takeuchi Y, Tamura Y, et al. Interaction between cancer and bone marrow cells induce osteoclast differentiation factor expression and osteoclast like cell formation in vitro. Biochem Biophys Res Commun. 2000 ; 267:632-637
81． Nagai M, Kyakumato S, Sato N. Cancer cell responsible for humoral hypercalcemia express mRNA encoding a secreted form of ODF/TRANCE that induces osteoclast formation. Biochem Biophys Res Commun. 2000 ; 269 : 532-536
82． Capparelli C, Kostenuik PJ, Morony S, et al. Osteoprotegerin prevents and reverses hypercalcemia in a murine model of humormal hypercalcemia of malignancy. Cancer Res. 2000 ; 60 : 783-787
83． Morony S, Capparelli C, Kostenuik PJ, et al. Osteoprotegerin prevents osteolytic bone destruction in both athymic and synergeneic models of experimental tumor metastasis to bone. J Bone Miner Res. 1999; 14(suppl 1) :1125
84． Honore P, Luger NM, Sabino MAC, et al. Osteoprotegerin blocks bone cancer induced skeletal destruction, skeletal pain and pain-ralated neurochemical reorganization of the spinal cord. Nat Med. 2000;5:521-528
85． Bucay N, Sarosi I.Dunstan CR, et al. Osteoprotegerin deficent mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998; 12:1260-1268
86． Min H, Morony S, Sarosi I, et al. Osteoprotegerin reverses osteoporosis by inhibiting endpsteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis. J Exp Med. 2000; 192:463-474