健骨二仙丸防治骨质疏松机理及一氧化氮调控机制研究
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摘要
“龟鹿二仙膏”为传统古方，出自《医便》，方用鹿胶、龟胶、人参、枸杞
四味，为阴阳气血交补之剂，可填阴补精，益气壮阳。根据导师多年相关学术临
床经验，在“龟鹿二仙膏”的基础上，以益气补肾为基本法则，研制了防治骨质
疏松药物“健骨二仙丸”胶囊。观察健骨二仙丸胶囊对骨质疏松症动物模型的治
疗效果和探讨其作用机制。
建立骨质疏松小鼠模型应用于中医药研究，用健骨二仙丸治疗卵巢切除小鼠
有明显的效果，骨密度升高，骨生物力学显著改善，血钙维持在正常水平，ALP
水平显著降低，在抑制骨吸收作用的同时并不引起明显子宫增生，在某些生物力
学指标方面优于后17β雌二醇组及Raloxifene组，弹性载荷、弹性桡度、最大载
荷等方面表现突出。明显抑制OVX小鼠体内骨吸收活动，而又同时具有
Raloxifene不刺激子宫增生等优点，这方面是17β雌二醇所不具有的。
给予健骨二仙丸治疗的卵巢切除大鼠骨密度上升，骨强度和骨韧性保持基本
较为完好，健骨二仙丸治疗骨质疏松症不仅在于其增加了骨钙含量，提高了骨量，
更在于其促进骨组织Ⅰ型胶原的合成与分泌，为羟基磷灰石提供了赖以沉积的支
架，并改善了骨组织的显微结构，从而提高了骨质量，使骨的生物力学性能增强，
骨折危险性降低。原位杂交、免疫组化实验显示健骨二仙丸可抑制OVX大鼠升
高的iNOS表达，对iNOS mRNA表达有明显的下调作用，提示健骨二仙丸可从
mRNA转录水平调控NOS的活性，从而减轻功能亢进的OC性骨吸收，而对骨
组织正常生理活动中的eNOS合成，有适度促进作用。健骨二仙丸还可促进骨髓
基质细胞向OB转化，可增强OB活动及功能，对OVX大鼠显著降低骨吸收的
同时，不抑制骨形成而起到调控或者促进作用，有利于骨组织的重建及骨量的恢
复，从而重建骨形成-吸收新平衡偶联。
健骨二仙丸对体外培养的人类成骨细胞株TE-85生长增殖有明显促进作用，
增加ALP、AGP的合成并释放，对细胞因子或AG作用下功能活动较低下的细
胞活性也可得到一定恢复。并在国内骨科率先应用FITC-Annexin V/PI双标记
广州中医药大学附间深训市中医赐 琴臼 阂土生导师 而劲夫
——
技术，通过流式细胞仪检测 TE 85细胞生存及凋亡情况，并结合 Bcl．2、Bax免
疫组化染色、iNOS含量测定等实验探讨健骨二仙丸防治骨质疏松及其调控一氧
化氮的可能机制。实验结果显示健骨二仙丸作用可总结为：促进生理情况下eNOS
表达，抑制病理状态下的iNOS产生；在转录，翻译水平对施加有利影响，可使
TE 85细胞Bcl毛表达增加而Bax产生下降，从而使成骨细胞在更长时间内保持
旺盛的功能状态。可降低凋亡细胞比例，减少骨细胞非正常生理状态下的凋亡，
延长其功能活动时间，从而促进骨重建。
以益气补肾为基本法则的健骨二仙丸不同于单纯激素替代疗法，作用不是单
一的，如调控细胞间信息传递途径、促进骨髓基质细胞向成骨细胞分化，增强成
骨细胞功能活动，延长其生存时间等，因而是多方面的多位点的集合作用，这种
药理作用是调动作用，调节作用，非激素替代作用，对骨质疏松的防治提供了有
效合理的途径。
Gui-Lu-Er-Xian-Gao" is a traditional Chinese prescription, replenishing Qi and
tonifying Yang, and nourishing Yin, base on my tutor clinic experience, replenishing
Qi and nourishing kidney deficiency is the foundation principle , we develop the
new traditional Chinese prescription "Jiang-Gu-Er-Xian- Wan" observed the
therapeutic efficacy for osteoporosis and discuse mechanism, such as nitric oxide, and
apoptosis.
1. The experiment of OVX mices.
The postmenopausal model was established by cutting off the bilateral ovaries of
the Kunming mices (10-11 weeks old). Baseline and sham-operated groups wer set up
at the same time. The mices were either sham-operated or ovariectomized(OVX)
besides one group was used as the base-line control. Three treated groups, received
either 17-f3 estradiol or Raloxifene, or "Jiang-Gu-Er-Xian- Wan" Bone mineral
density(BMD), bone biomechanicanics, biochemical were tested.
The mice in OVX group got the following biological changes: the body weight
increased with the fastest speed; the uteruses withered; the bone mineral density?and
mineral content decreased greatly; biomechanical and histomorphological parameters
lowered down, and turnover of bone accelerated compared with sham group.
After treated with "Jiang-Gu-Er-Xian-Wan" the bone mineral density of the mice
restored significantly and almost reach the level of sham operated group. Other
parameters were also improved greatly, mineral content increased, biomechanical
restored, and blood ALP were lowered down comparing with OVX group, calcium,
phosphorus levels is in norml range. The uteruses were some proliferated, but at a
lower degree, just like the Raloxifene not stimulate the uteruses, contrast to 17
-estrodial group, the uteruses appeared hyperplasia (p <0.01).
2. The experiment of OVX rats.
Three-month-old female wistar rats were randomly divided into five groups. The
rats were either sham-operated or ovariectomized(OVX) besides one group was used
as the base-line control. Three treated groups, received either Premarin or low dose of
herbs, or high dose of herbs. Bone mineral density(BMD), bone biomechanicanics,
BGP, biochemical were tested. And the lumbar vertebrae section is stud by
hybridization in situ of type I collagen, INOS, immunohistochemistry of
iNOS, eNOS, and RT-PCR for iNOS.
Rats in OVX group got the following changes: weight increased, BMD decreased,
uterus withered, biomechanical and histomorphological parameters lowered down,
and turnover of bone accelerated compared with sham group.
After treated with 揓iang-Gu-Er-Xian-Wan? bone mineral density was restored
greatly, biomechanical parameters were greatly improved, bone turnover was reduced.
But uterus didn抰 appear hyperplasia, nor did blood calcium or blood phosphorus level
increased.
Hybridization in situ , immunohistochemistry and RT桺CR show the iNOS
protein is expressed strongly in the OVX group, show the NO play a role in
stimulate OC bone absorption. Premarin group iNOS is lighten in some degree,
Jiang-Gu-Er-Xian-Wan?group the iNOS is barely detectable, but the eNOS
expression is strength, the hybridization in situ of type I collagen is strongly
expressed.
3. The experiment of HOS TE 85 cell strain.
Recent work has shown that nitric oxide (NO) induction by nitric oxide
synthase(NOS) is physiological mediator of bone cell function and demonstrate that it
may be poss
四味，为阴阳气血交补之剂，可填阴补精，益气壮阳。根据导师多年相关学术临
床经验，在“龟鹿二仙膏”的基础上，以益气补肾为基本法则，研制了防治骨质
疏松药物“健骨二仙丸”胶囊。观察健骨二仙丸胶囊对骨质疏松症动物模型的治
疗效果和探讨其作用机制。
建立骨质疏松小鼠模型应用于中医药研究，用健骨二仙丸治疗卵巢切除小鼠
有明显的效果，骨密度升高，骨生物力学显著改善，血钙维持在正常水平，ALP
水平显著降低，在抑制骨吸收作用的同时并不引起明显子宫增生，在某些生物力
学指标方面优于后17β雌二醇组及Raloxifene组，弹性载荷、弹性桡度、最大载
荷等方面表现突出。明显抑制OVX小鼠体内骨吸收活动，而又同时具有
Raloxifene不刺激子宫增生等优点，这方面是17β雌二醇所不具有的。
给予健骨二仙丸治疗的卵巢切除大鼠骨密度上升，骨强度和骨韧性保持基本
较为完好，健骨二仙丸治疗骨质疏松症不仅在于其增加了骨钙含量，提高了骨量，
更在于其促进骨组织Ⅰ型胶原的合成与分泌，为羟基磷灰石提供了赖以沉积的支
架，并改善了骨组织的显微结构，从而提高了骨质量，使骨的生物力学性能增强，
骨折危险性降低。原位杂交、免疫组化实验显示健骨二仙丸可抑制OVX大鼠升
高的iNOS表达，对iNOS mRNA表达有明显的下调作用，提示健骨二仙丸可从
mRNA转录水平调控NOS的活性，从而减轻功能亢进的OC性骨吸收，而对骨
组织正常生理活动中的eNOS合成，有适度促进作用。健骨二仙丸还可促进骨髓
基质细胞向OB转化，可增强OB活动及功能，对OVX大鼠显著降低骨吸收的
同时，不抑制骨形成而起到调控或者促进作用，有利于骨组织的重建及骨量的恢
复，从而重建骨形成-吸收新平衡偶联。
健骨二仙丸对体外培养的人类成骨细胞株TE-85生长增殖有明显促进作用，
增加ALP、AGP的合成并释放，对细胞因子或AG作用下功能活动较低下的细
胞活性也可得到一定恢复。并在国内骨科率先应用FITC-Annexin V/PI双标记
广州中医药大学附间深训市中医赐 琴臼 阂土生导师 而劲夫
——
技术，通过流式细胞仪检测 TE 85细胞生存及凋亡情况，并结合 Bcl．2、Bax免
疫组化染色、iNOS含量测定等实验探讨健骨二仙丸防治骨质疏松及其调控一氧
化氮的可能机制。实验结果显示健骨二仙丸作用可总结为：促进生理情况下eNOS
表达，抑制病理状态下的iNOS产生；在转录，翻译水平对施加有利影响，可使
TE 85细胞Bcl毛表达增加而Bax产生下降，从而使成骨细胞在更长时间内保持
旺盛的功能状态。可降低凋亡细胞比例，减少骨细胞非正常生理状态下的凋亡，
延长其功能活动时间，从而促进骨重建。
以益气补肾为基本法则的健骨二仙丸不同于单纯激素替代疗法，作用不是单
一的，如调控细胞间信息传递途径、促进骨髓基质细胞向成骨细胞分化，增强成
骨细胞功能活动，延长其生存时间等，因而是多方面的多位点的集合作用，这种
药理作用是调动作用，调节作用，非激素替代作用，对骨质疏松的防治提供了有
效合理的途径。
Gui-Lu-Er-Xian-Gao" is a traditional Chinese prescription, replenishing Qi and
tonifying Yang, and nourishing Yin, base on my tutor clinic experience, replenishing
Qi and nourishing kidney deficiency is the foundation principle , we develop the
new traditional Chinese prescription "Jiang-Gu-Er-Xian- Wan" observed the
therapeutic efficacy for osteoporosis and discuse mechanism, such as nitric oxide, and
apoptosis.
1. The experiment of OVX mices.
The postmenopausal model was established by cutting off the bilateral ovaries of
the Kunming mices (10-11 weeks old). Baseline and sham-operated groups wer set up
at the same time. The mices were either sham-operated or ovariectomized(OVX)
besides one group was used as the base-line control. Three treated groups, received
either 17-f3 estradiol or Raloxifene, or "Jiang-Gu-Er-Xian- Wan" Bone mineral
density(BMD), bone biomechanicanics, biochemical were tested.
The mice in OVX group got the following biological changes: the body weight
increased with the fastest speed; the uteruses withered; the bone mineral density?and
mineral content decreased greatly; biomechanical and histomorphological parameters
lowered down, and turnover of bone accelerated compared with sham group.
After treated with "Jiang-Gu-Er-Xian-Wan" the bone mineral density of the mice
restored significantly and almost reach the level of sham operated group. Other
parameters were also improved greatly, mineral content increased, biomechanical
restored, and blood ALP were lowered down comparing with OVX group, calcium,
phosphorus levels is in norml range. The uteruses were some proliferated, but at a
lower degree, just like the Raloxifene not stimulate the uteruses, contrast to 17
-estrodial group, the uteruses appeared hyperplasia (p <0.01).
2. The experiment of OVX rats.
Three-month-old female wistar rats were randomly divided into five groups. The
rats were either sham-operated or ovariectomized(OVX) besides one group was used
as the base-line control. Three treated groups, received either Premarin or low dose of
herbs, or high dose of herbs. Bone mineral density(BMD), bone biomechanicanics,
BGP, biochemical were tested. And the lumbar vertebrae section is stud by
hybridization in situ of type I collagen, INOS, immunohistochemistry of
iNOS, eNOS, and RT-PCR for iNOS.
Rats in OVX group got the following changes: weight increased, BMD decreased,
uterus withered, biomechanical and histomorphological parameters lowered down,
and turnover of bone accelerated compared with sham group.
After treated with 揓iang-Gu-Er-Xian-Wan? bone mineral density was restored
greatly, biomechanical parameters were greatly improved, bone turnover was reduced.
But uterus didn抰 appear hyperplasia, nor did blood calcium or blood phosphorus level
increased.
Hybridization in situ , immunohistochemistry and RT桺CR show the iNOS
protein is expressed strongly in the OVX group, show the NO play a role in
stimulate OC bone absorption. Premarin group iNOS is lighten in some degree,
Jiang-Gu-Er-Xian-Wan?group the iNOS is barely detectable, but the eNOS
expression is strength, the hybridization in situ of type I collagen is strongly
expressed.
3. The experiment of HOS TE 85 cell strain.
Recent work has shown that nitric oxide (NO) induction by nitric oxide
synthase(NOS) is physiological mediator of bone cell function and demonstrate that it
may be poss
引文
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48． Malluche HH, Matthews C, Faugere MC, Fanti P, Endres DB, Freidler RM. l-25dihydroxyvitamin D maintains bone cell activity, and parathyroid hormone modulates bone cell number in dogs.
49． Turner CH, Sato M, Bryant HU. Raloxifene preserves bone strength and bone mass in ovariectomized rats. Endocrinology, 1994, 135(6) : 2001-2005．
50． Rahimian R, Laher I, Dube G, et al . Estrogen and selective estrogen receptor modulator LY117018 enhance release of nitric oxide in rat aorta[J]. J Pharmacol Exp Ther, 1997 , 283(1) : 116-122．
51． Jones CD, Jevnikar MG, Pike AJ, etal. Antiestrogens. Structure-activity studies in a series of 3-Kroyl-2-arylbenzo[b]thiophene derivatives leading to [6-hydroxy-2 (4-hydroxyphenyl) benzo [b]thien-3-yl][4-[2-(1-piperidinyl) ethoxy]-phenyl] menthanone hydrochloride(LY1
56758), a remarkablely effective estrogen antagonist with only minimal intrinsic estrogenicity. JMed Chem, 1984,27(3): 105-109.
52. SatoM ,RippyMK,BryantHU. Raloxifene, tamoxifen, nafoxidine, or Estrogen effects on reproductive and non reproductive tissues in Ovariectomized rats. FASEBJ, 1996, 10: 905-912.
53. Frolik CA, Bryant HU,Black EC, et al. Time dependent changes in biochemical bone markers ands erum cholesterol in ovariectomized rats: effects of raloxifene HCl, tamoxifen, estrogen, and alendronate. Bone, 1996, 18(2): 621-627.
54. Delmas PD, Bjarnason NH, Mitlak BH, et al.Effects of raloxifene on bone mineral density, serum cholesterolconcen-trations,and uterine endometrium in postmenopausal women. N Eng J Med, 1997, 337(23): 1641-1648.
55. Hua Zhu KE, Hollis A. Simmons, Christine M. Pirie, et al. Droloxifene, a new estrogen antagonist/agonist, prevents bone loss in ovariectomized rats. Endocrinology. 1995;136: 2435-41.
56．胡侦明，戴克戎、朱汉民．卵巢切除对鼠及异常应力鼠骨密度影响的比较研究．中华骨科杂志．1998；18，9：555-558．
57. Adrienne M. Flanagan and Timothy J. Chambers. Inhibition of bone resorption by bisphosphonates: interactions between bisphosphonaes, osteoclasts, and bone. Calcif Tissue Int. 1991; 49: 407-415.
58．崔伟，刘成林．基础骨生物力学(一)，中国骨质疏松杂志．1997，3(4)：82-85．
59．崔伟，刘成林．基础骨生物力学(一)，中国骨质疏松杂志．1998，4(1)：90-92．
60. B. C. Toolan, M. Shea, E. R. Myers, R. E. Borchers, et al. Effects of 4-amino-l-hydroxybutylidene bisphosphonate on bone biomechanics in rats. J Bone Miner Res. 1992; 7, 12: 1399-1406.
61. Kuhn J L, Goldstein S A, Choi K. Comparison of the trabecular and cortical tissue moduli from human iliac crests. J Orthop Res, 1989, 7: 876-881.
62. Matin B, Ishida J. The relative effects of cillagen fiber orientation, proosity, denity and mineralization on bone strength. J Biomech, 1989, 22:419-423
63．薛延，骨质疏松症诊断与治疗指南．北京：科学出版社．1999，35-45．
64． Pacifici R.ls there a causal role of IL-1 in postmenopausal bone loss? Calcif Tissue Int. 1992, 50: 295-299．
65． Kimble RB,Vannice JL,Bloedow DC, et al. Jnterleukin-1 receptor antagonist decreases bone loss and bone resorption in ovariectomized rats. J Clin Invest, 1994, 93: 1959-1965．
66． Pacifici R,Brown C,Puscheck E,et al.The effects of surgical menopause and estrogen replacement on cytokine release from human blood monocytes. Proc Natl Acad Sci USA, 1991,88:5134-5139．
67． Ralston SH,Russel RGG,Gowen M. Estrogen inhibits release of tumor necrosis factor from peripheral blood mononuclear cells in postmenopausal women. J Bone Miner Res, 1990, 5: 983-986．
68． Komm BS.Estrogen binding receptor mRNA and biologic response in osteoblast-like osteosarcoma cells.Science, 1988,241:81-84．
69． Oursle MJ,Osdoby P,Pyfferoen J,et al.Avian osteoclast as estrogen target cells.Proc Natl Acad Sci USA,1991,88:6613-6617．
70． Mano H, Yuasa T, Kameda T. et al. Mammalian mature osteoclast as estrogen target cells.Biochem Biophys Res Commun,1996, 223: 637-642．
71． Etienne M C, Fischel J L, Milano G. Steroid receptors in human osteoblast-like cells. Eur J Cancer, 1990,26:807-810．
72． Masuyama A, Ouchi Y, Sago F, et al. Characteristics of steroid hormone receptors in cultured HOS TE 85 osteoblastic cells and effect of steoid hormones on cell proliferation,Calcif. Tissue Int. 1992,51:376-38
73． Gunnet JW, Granger K, Cryan E, et al. Characterization of the progestin receptors in the human TE85 and murine MC3T3-E1 osteoblast-like cell lines. J Endocrinol ,1999, 163(10) :139-47．
74． Macpherson H, noble B S, ralston S H. Expression and functional role of nitric oxide synthase isoforms in human osteoblast-like cells. Bone, 1999, 24(3) : 179-185．
75． Thomas AO, Michael A, Victoria S, et al. Progressive development of the rat osteoblast phenotype in vitro : reciprocal relationship in expression of genesas sociated with osteoblast proliferation and differentiation during formation of the bone extracelluar matrix. J Cell Physiol, 1990, 143:420-425．
76. Tomkinson A, Gevers EF, Wit JM, et al.The role of estrogen in the control of rat osteocyte apoptosis. J Bone Miner Res, 1998, 13: 1243-1250.
77. Nicoletti l, Migliorati G, Pagliacci MC. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods, 1991,139: 2712-2718.
78. Fadok V A, Voelker D R, Campbell P A, et al. Exposure of phosphatidyl serine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophage. J Immuol, 1992;148:220-225.
79. Vermes I, Haanen C, Steffens-Nakken H, et al. A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptosis cells using fluorescein labeled Annexin V. J Immunol Methods, 1995,184: 393-399.
80. PengLM, LiuJJ.A novel method for quantita tivean alysis of apoptosis. Lab Inves, 1997, 77: 547-555.
81. Sarkar D, Nagaya T, Koga K, et al. Culture in vector-averaged gravity under clinostat rotation results in apoptosis of osteoblastic ROS 17/2.8 cells, J Bone Miner Res, 2000 15(3): 489-98.
82. Stanislaus D, Yang X, Liang JD,et al. In vivo regulation of apoptosis in metaphyseal trabecular bone of young rats by synthetic human parathyroid hormone (1-34) fragment. Bone, 2000,27(8):209-18.
83．萧劲夫，李昂．中医药治疗男性骨质疏松及其股骨颈骨折．中国骨质疏松杂志，1997，(3)：46-47．
84．李昂．先秦伤科史略．中华医史杂志，1996，(1)：18-22．
85．萧劲夫．岭南正骨精要．广州：广东高教出版社．1995：64-66．
86．李芳芳，李恩，宋士军，等．补肾方剂及不同分离组分对成骨细胞增殖分化的影响．中国骨质疏松杂志，1998，4(3)：71-74．
87．胡梅．肾虚证大鼠骨胶原与钙含量的变化及其与肾主骨的关系．中国医药学报，1993，8(3)：48-52．
88．安胜军，李恩，赵京山．补肾方药对地塞米松所致实验性骨质疏松大鼠卵巢功能的影响．中国中西医结合杂志，2000，20(1)：46-48．
89．刘和娣，李思，佟晓旭，等．补肾中药对地塞米松诱发的骨质疏松大鼠体内雌激素和1，25(OH)_2D_3的影响．中国中西医结合杂志，1993，13(9)：544-545．
90．俞瑾，陈红英，王秋芝，等．肾主生殖的实验研究．中西医结合杂志1989；9(9)：548-551
2. 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 osteoporosis. Maturitas, 1997,26(1): 63-71.
3. Kimble RB, Vannice JL, Bloedow DC, et al. Interleukin-1 receptor antagonist decreases bone loss and bone resorption in ovariectomized rats. J Clin Invest, 1994, 93(5): 1959-67.
4. Ralston SH. Analysis of gene expression in human bone biopsies by polymerase chain reaction: evidence for enhanced cytokine expression in postmenopausal osteoporosis. J Bone Miner Res, 1994, 9(6): 883-90.
5. Kimble RB, Matayoshi AB, Vannice JL, et al. Simultaneous block of interleukin-1 and tumor necrosis factor is required to completely prevent bone loss in the early postovariectomy period. Endocrinology, 1995, 136(7): 3054-61.
6．刘景生．细胞信息与调控．北京：北京医科大学/中国协和医科大学联合出版社，1998，207-228．
7. Maclntyre I,Zaida M,Alam ASMT, et al. Osteoclastic inhibition: An action of nitric oxide not mediated by cycle GMP. Proc Nati Acad Sci USA,1991,88:2936-294.
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14． Ueno M, Fukuda K, Oh M, et al. Protein kinase C modulates the synthesis of nitric oxide by osteoblasts. Calcif Tissue Int, 1998,63(1) : 22-26．
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16． Kioldrung MB. Skeletal tissue response to cytokines. Clin Orthop, 1990 ,258(3) : 245-278．
17． Ralston SH, Ho LP, Helfrich MH, et al. Nitric oxide: A cytokine-induced regulator of bone resorption. J Bone Miner Res, 1995, 10(7) : 1040-1044．
18． MacPherson H, Noble BS, Ralston SH, et al. Expression and functional role of nitric oxide synthase isoforms in human osteoblast-like cells. Bone, 1999, 24(3) : 179-185．
19． Ralston SH, Todd D, Helfrich MH, et al. Human osteoblastlike cells produce nitric oxide and express inducible nitric oxide synthase. Endocrinology. 1994, 135(1) : 330-336．
20． Lowik CW, Nibbering PH, van de Ruit M, et al. Indu-cible production of nitric oxide in osteoblast like cells and in fetal bone explants is associated with supression of osteoclastic bone resorption. J Clin Invest, 1994, 93(4) :1465-1472．
21． Damoulis PD, Hauschka PV. Nitric oxide acts in conjunction with proinflammatory cytokines to promote cell death in osteoblasts. J Bone Miner Res, 1997, 12(3) : 412-422．
22． Kanematsu M, Ikeda K, Yamada Y, et al. Interaction between nitric oxide synthase and cyclooxygenase pathways in osteoblastic MC3T3-E1 cells. J Bone Miner Res, 1997, 12(11) : 1789-1796．
23． McAllister TN, Frangos JA. Steady and transient fluid shear stress stimulate NO release in osteoblasts through distinct biochemical pathways. J Bone Miner Res, 1999 , 14(6) :930-936．
24． Wimalawansa SJ, De Marco G, Gangula P, et al. Nitric oxide donor alleviates ovariectomy-induced bone loss. Bone, 1996, 18(4) : 301-304．
25． Wima SJ, Chapa MT, Yalla C,et al. Prevention of corticosteroid-induced bone loss with nitric oxide donor nitroglycerin in male rats. Bone, 1997,21(3) : 275-279．
26． Fox SW, Chambers TJ, Chow JW. Nitric oxide is an early mediator of the increase in bone formation by mechanical stimulation. Am J Physiol ,1996, 270(6) : 955-960．
27． Gray TK, Flynn TC, Gray KM, et al. 17 β-estradiol acts directly on the clonal osteoblastic cell line UMR 106． Proc Natl Acad Sci U S A, 1987,84(17) 6267-6271．
28． Hughes FJ, Buttery LD, Hukkanen MV, et al. Cytokine induced prostaglandin E2 synthesis and cyclooxygenase-2 activity are regulated both by a nitric oxide dependent and independent mechanism in rat osteobl-asts in vitro. J Biol Chem, 1999,274(3) : 1776-1781．
29． Simoncini T, Genazzani AR. Raloxifene acutely stimulates nitric oxide release from human endothelial cells via an activation of endothelial nitric oxide synthase. J Clin Endocrinol Metab , 2000, 85(8) : 2966-2969．
30． Stacey E, Korkia P, Hukkanen MV, et al. Decreased nitric oxide levels and bone turnover in amenorrheic athletes with spinal osteopenia. J Clin Endocrinol Metab ,1998, 83(9) : 3056-3060．
31． Pitsillides A A, Rawlinson SC, Suswillo RF, et al. Mechanical strain-induced NO production by bone cells: a possible role in adaptive bone remodeling. FASEB J, 1995, 9(9) : 1614-1622．
32． 邓廉夫。骨质疏松发生的细胞学基础及其防治.实用老年医学.2000,14(3) :119-12．
33． Owen ME. Lineage of osteogenic cells and their relationship to the stromal system. J Bone Miner Res, 1994． 9(7) : 1984． 1-25
34． Oltvai ZN,Milliman CL and Korsmeyer SJ.Bcl-2 heterodimerizes in vivo with a conserved homolog,Bax that accelerates programmed cell death Cell,1993,74:609
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36． Nable B S, Stevens H, Loveridge N, et al. Identification of apoptotic changes in osteocytes in normal and pathological human bone.Bone,1997,20(3) :273-282．
37． Txanaka K.Matsuo T,Ohta M, et al.time-Iapse microcinematography of osteocytes. Miner Electrolyte Metab, 1995,21(1) :189-192．
38． Elmardi A S,Katchburian M V,Katchburian E. Electron microscopy of developing calvaria reveals images that sugguest that osteoclasts engulf and destroy osteocytes during bone resorption.Calcif Tissue Int,1990, 46(l):239-245．
39． Klein RF,Fausti KA,Carlos AS, et al.Ethanol inhibits human osteoblastic cell proliferation.. Alcohol Clin Exp Res, 1996,20(3) :572-578
40． Ookawa K,Tisuchida S,Adachi J, et al.Differentiation induced by RB expression and apoptosis induced by p53 expression in an osteosarcoma cell line. Oncogene,l997,14(12) : 1389-1396．
41． Mano H,Yuasa .T,Kameda K, et al.Mammalian mature osteoclasts as estrogen taarget cells.Biochem Res,1996,223(4) :637-642．
42． Hughes DE,Dai A,Tifee JC, et al.Estrogen promotes apoptosis of murine osteoclasts mediated by TGF. Nat Med, 1996,2(10) : 1132-1136．
43． Kameda T,Mano H,Yuasa T, et al.Estrogen inhibits bone resorption by directly inducing opoptosis of the bone resorbing osteoclasts. J Exp Med, 1997, 186(4) : 489-495．
44． Van't T,Ralston SH.Cytokine induced nitric oxide inhibits bone resorption by inducing apoptosis of osteoclast progenitors and suppressing osteoclast activity.Bone Miner Res,1997,12(12) : 1797-802．
45． Dempster DW,Moonga BS,Stein LS, et al.Glucocorticoids inhibits bone resorption by isolated osteoclasts by enhancing apoptosis. J Endocrinol, 1997,154(3) : 397-406．
46． 董玉峰.细胞凋亡与骨重建.中国矫形外科杂志,2000,7(9) :34-38．
47． Spiess YH, Price PA, Deftos JLL, Manolagas SC. Phenotype-associated changes in the effects of l-25dihydroxyvitamin D3 on alkaline phosphatase and bone Gla-protein of rat ostoblastic cells. Endocrinology, 1986 118:1340-1346
48． Malluche HH, Matthews C, Faugere MC, Fanti P, Endres DB, Freidler RM. l-25dihydroxyvitamin D maintains bone cell activity, and parathyroid hormone modulates bone cell number in dogs.
49． Turner CH, Sato M, Bryant HU. Raloxifene preserves bone strength and bone mass in ovariectomized rats. Endocrinology, 1994, 135(6) : 2001-2005．
50． Rahimian R, Laher I, Dube G, et al . Estrogen and selective estrogen receptor modulator LY117018 enhance release of nitric oxide in rat aorta[J]. J Pharmacol Exp Ther, 1997 , 283(1) : 116-122．
51． Jones CD, Jevnikar MG, Pike AJ, etal. Antiestrogens. Structure-activity studies in a series of 3-Kroyl-2-arylbenzo[b]thiophene derivatives leading to [6-hydroxy-2 (4-hydroxyphenyl) benzo [b]thien-3-yl][4-[2-(1-piperidinyl) ethoxy]-phenyl] menthanone hydrochloride(LY1
56758), a remarkablely effective estrogen antagonist with only minimal intrinsic estrogenicity. JMed Chem, 1984,27(3): 105-109.
52. SatoM ,RippyMK,BryantHU. Raloxifene, tamoxifen, nafoxidine, or Estrogen effects on reproductive and non reproductive tissues in Ovariectomized rats. FASEBJ, 1996, 10: 905-912.
53. Frolik CA, Bryant HU,Black EC, et al. Time dependent changes in biochemical bone markers ands erum cholesterol in ovariectomized rats: effects of raloxifene HCl, tamoxifen, estrogen, and alendronate. Bone, 1996, 18(2): 621-627.
54. Delmas PD, Bjarnason NH, Mitlak BH, et al.Effects of raloxifene on bone mineral density, serum cholesterolconcen-trations,and uterine endometrium in postmenopausal women. N Eng J Med, 1997, 337(23): 1641-1648.
55. Hua Zhu KE, Hollis A. Simmons, Christine M. Pirie, et al. Droloxifene, a new estrogen antagonist/agonist, prevents bone loss in ovariectomized rats. Endocrinology. 1995;136: 2435-41.
56．胡侦明，戴克戎、朱汉民．卵巢切除对鼠及异常应力鼠骨密度影响的比较研究．中华骨科杂志．1998；18，9：555-558．
57. Adrienne M. Flanagan and Timothy J. Chambers. Inhibition of bone resorption by bisphosphonates: interactions between bisphosphonaes, osteoclasts, and bone. Calcif Tissue Int. 1991; 49: 407-415.
58．崔伟，刘成林．基础骨生物力学(一)，中国骨质疏松杂志．1997，3(4)：82-85．
59．崔伟，刘成林．基础骨生物力学(一)，中国骨质疏松杂志．1998，4(1)：90-92．
60. B. C. Toolan, M. Shea, E. R. Myers, R. E. Borchers, et al. Effects of 4-amino-l-hydroxybutylidene bisphosphonate on bone biomechanics in rats. J Bone Miner Res. 1992; 7, 12: 1399-1406.
61. Kuhn J L, Goldstein S A, Choi K. Comparison of the trabecular and cortical tissue moduli from human iliac crests. J Orthop Res, 1989, 7: 876-881.
62. Matin B, Ishida J. The relative effects of cillagen fiber orientation, proosity, denity and mineralization on bone strength. J Biomech, 1989, 22:419-423
63．薛延，骨质疏松症诊断与治疗指南．北京：科学出版社．1999，35-45．
64． Pacifici R.ls there a causal role of IL-1 in postmenopausal bone loss? Calcif Tissue Int. 1992, 50: 295-299．
65． Kimble RB,Vannice JL,Bloedow DC, et al. Jnterleukin-1 receptor antagonist decreases bone loss and bone resorption in ovariectomized rats. J Clin Invest, 1994, 93: 1959-1965．
66． Pacifici R,Brown C,Puscheck E,et al.The effects of surgical menopause and estrogen replacement on cytokine release from human blood monocytes. Proc Natl Acad Sci USA, 1991,88:5134-5139．
67． Ralston SH,Russel RGG,Gowen M. Estrogen inhibits release of tumor necrosis factor from peripheral blood mononuclear cells in postmenopausal women. J Bone Miner Res, 1990, 5: 983-986．
68． Komm BS.Estrogen binding receptor mRNA and biologic response in osteoblast-like osteosarcoma cells.Science, 1988,241:81-84．
69． Oursle MJ,Osdoby P,Pyfferoen J,et al.Avian osteoclast as estrogen target cells.Proc Natl Acad Sci USA,1991,88:6613-6617．
70． Mano H, Yuasa T, Kameda T. et al. Mammalian mature osteoclast as estrogen target cells.Biochem Biophys Res Commun,1996, 223: 637-642．
71． Etienne M C, Fischel J L, Milano G. Steroid receptors in human osteoblast-like cells. Eur J Cancer, 1990,26:807-810．
72． Masuyama A, Ouchi Y, Sago F, et al. Characteristics of steroid hormone receptors in cultured HOS TE 85 osteoblastic cells and effect of steoid hormones on cell proliferation,Calcif. Tissue Int. 1992,51:376-38
73． Gunnet JW, Granger K, Cryan E, et al. Characterization of the progestin receptors in the human TE85 and murine MC3T3-E1 osteoblast-like cell lines. J Endocrinol ,1999, 163(10) :139-47．
74． Macpherson H, noble B S, ralston S H. Expression and functional role of nitric oxide synthase isoforms in human osteoblast-like cells. Bone, 1999, 24(3) : 179-185．
75． Thomas AO, Michael A, Victoria S, et al. Progressive development of the rat osteoblast phenotype in vitro : reciprocal relationship in expression of genesas sociated with osteoblast proliferation and differentiation during formation of the bone extracelluar matrix. J Cell Physiol, 1990, 143:420-425．
76. Tomkinson A, Gevers EF, Wit JM, et al.The role of estrogen in the control of rat osteocyte apoptosis. J Bone Miner Res, 1998, 13: 1243-1250.
77. Nicoletti l, Migliorati G, Pagliacci MC. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods, 1991,139: 2712-2718.
78. Fadok V A, Voelker D R, Campbell P A, et al. Exposure of phosphatidyl serine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophage. J Immuol, 1992;148:220-225.
79. Vermes I, Haanen C, Steffens-Nakken H, et al. A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptosis cells using fluorescein labeled Annexin V. J Immunol Methods, 1995,184: 393-399.
80. PengLM, LiuJJ.A novel method for quantita tivean alysis of apoptosis. Lab Inves, 1997, 77: 547-555.
81. Sarkar D, Nagaya T, Koga K, et al. Culture in vector-averaged gravity under clinostat rotation results in apoptosis of osteoblastic ROS 17/2.8 cells, J Bone Miner Res, 2000 15(3): 489-98.
82. Stanislaus D, Yang X, Liang JD,et al. In vivo regulation of apoptosis in metaphyseal trabecular bone of young rats by synthetic human parathyroid hormone (1-34) fragment. Bone, 2000,27(8):209-18.
83．萧劲夫，李昂．中医药治疗男性骨质疏松及其股骨颈骨折．中国骨质疏松杂志，1997，(3)：46-47．
84．李昂．先秦伤科史略．中华医史杂志，1996，(1)：18-22．
85．萧劲夫．岭南正骨精要．广州：广东高教出版社．1995：64-66．
86．李芳芳，李恩，宋士军，等．补肾方剂及不同分离组分对成骨细胞增殖分化的影响．中国骨质疏松杂志，1998，4(3)：71-74．
87．胡梅．肾虚证大鼠骨胶原与钙含量的变化及其与肾主骨的关系．中国医药学报，1993，8(3)：48-52．
88．安胜军，李恩，赵京山．补肾方药对地塞米松所致实验性骨质疏松大鼠卵巢功能的影响．中国中西医结合杂志，2000，20(1)：46-48．
89．刘和娣，李思，佟晓旭，等．补肾中药对地塞米松诱发的骨质疏松大鼠体内雌激素和1，25(OH)_2D_3的影响．中国中西医结合杂志，1993，13(9)：544-545．
90．俞瑾，陈红英，王秋芝，等．肾主生殖的实验研究．中西医结合杂志1989；9(9)：548-551
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