Association Between Repeat Length of Exon 1 CAG Microsatellite in the Androgen Receptor and Bone Density in Men is Modulated by Sex Hormone Levels

In this study we examined whether the androgen receptor (AR) gene CAG repeat polymorphism and serum androgen levels are associated with bone mineral density (BMD) and changes in BMD during 2–3 years in 229 healthy men 41–76 years old. Microsatellite analysis was performed on an automated sequencer. Indices of bioavailable testosterone (free testosterone [FT] and free androgen index) were calculated. BMD was measured using both dual-energy X-ray absorptiometry and quantitative ultrasound. All participants completed a questionnaire regarding major possible osteoporosis risk factors. In linear regression analysis there was a modest positive association, which was independent of age and body mass index (BMI), between AR repeat length and BMD at all sites. Although this association was significant independent of BMI, analyses in the subgroup of obese men (BMI > 30) did not reach significance, while the effect was enhanced when analyzing only nonobese men (BMI ≤ 30). There was no association between the AR gene polymorphism and rate of bone loss, FT, and BMD or testosterone and bone loss. Interestingly, the association between AR and BMD was modified by total testosterone. The lowest age- and BMI-adjusted average femoral neck BMD was found among men in the lowest tertile for both AR repeat length and FT, whereas men within the higher categories of these variables displayed the highest BMD. In conclusion, there is a positive association between the AR CAG repeat polymorphism and BMD, which is modified by androgen levels in healthy men.     

IGF-I, GH, and Sex Steroid Effects in Normal Mammary Gland Development

Although the pubertal surge of estrogen is the immediate stimulus to mammary development, the action of estrogen depends upon the presence of pituitary growth hormone and the ability of GH to stimulate production of IGF-I in the mammary gland. Growth hormone binds to its receptor in the mammary fat pad, after which production of IGF-I mRNA and IGF-I protein occurs. It is likely that IGF-I then works through paracrine means to stimulate formation of TEBs, which then form ducts by bifurcating or trifurcating and extending through the mammary fat pad. By the time pubertal development is complete a tree-like structure of branching ducts fills the rodent mammary fat pad. In addition to requiring IGF-I in order to act, estradiol also directly synergizes with IGF-I to enhance formation of TEBs and ductal morphogenesis. Together they increase IRS-1 phosphorylation and cell proliferation, and inhibit apoptosis. In fact, the entire process of ductal morphogenesis, in oophorectomized IGF-I^(−/−) knockout female mice, can occur as a result of the combined actions of estradiol and IGF-I. IGF-I also permits progesterone action in the mammary gland. Together they have been shown to stimulate a form of ductal morphogenesis, which is anatomically different from the kind induced by IGF-I and estradiol. Although both progesterone and estradiol synergize with IGF-I by increasing IGF-I action parameters, there must be other, as yet unknown mechanisms that account for the anatomical differences in the different forms of ductal morphogenesis observed (hyperplasia in response to IGF-I plus estradiol and single layered ducts in response to IGF-I plus progesterone). From the Bunnie Joan Sachs Laboratory, VA Medical Center, New York, NY, USA Supported in part by grants from DOD W81XWH-07-1-0488 and the Foundation for Growth and Endocrinology     

Differential effects of parathyroid hormone on protein phosphorylation in two osteoblastlike cell populations isolated from neonatal mouse calvaria

Summary To further understand the mechanism of PTH effects on bone and bone cells, we have analyzed the effect of PTH on specific protein phosphorylation in cells isolated from neonatal mouse calvaria. Four populations of cells (I–IV), isolated by sequential digestion with chromatographically purified bacterial collagenase isozymes and neutral proteinase, were cultured overnight. Alkaline phosphatase activity was greater than acid phosphatase activity in all four populations. PTH stimulated cyclic AMP production in all four populations, although the effect was greatest in populations II and III. Cultured cells were treated with PTH for up to 15 minutes. Cytosolic and membrane fractions were obtained and assayed forin vitro protein phosphorylation. No hormonal effects were found in membrane fractions. In cytosol fractions, treatment of the population II cells for 10–15 minutes with 0.1 μM PTH decreased the subsequent protein phosphorylation of an 85,000 M_r protein. In contrast, PTH treatment increasedin vitro phosphorylation of both the 85,000 and 35,000 M_r proteins in population III cells. Phosphorylation of the 35,000 M_r protein was cyclic AMP-dependent. All of the phosphoproteins appeared to be phosphorylated solely on serine or threonine residues except the 85,000 M_r protein which may also contain significant amounts of phosphotyrosine. Therefore, some of the effects of PTH are cyclic AMP-mediated and other effects may be mediated through tyrosine phosphorylation. These data indicate that PTH has differential effects onin vitro protein phosphorylation in two separable populations of isolated neonatal mouse calvarial cells and support a hypothesis that multiple osteoblastlike cells existin vivo.     

Vertebral bone resorptionin vitro: Effects of parathyroid hormone,calcitonin, 1,25 dihydroxyvitamin D3, epidermal growth factor,prostaglandin E2, and estrogen

Summary We have developed and characterized a new bone resorption system to test the effect of estrogen on vertebral bonein vitro. Neonatal mouse vertebral bones prelabeled with⁴⁵Ca were maintained in stationary tissue culture for 60–108 hours at 37°C in 5% CO₂/air. Each vertebral bone measured approximately 1 mm×1 mm. Hormonal treatments were added directly to the incubation medium. The morphological appearance of these bones, before and after the onset of resorption, was examined by scanning electron microscopy. Bone resorption was measured by determining the % of the bone⁴⁵Ca released into the incubation medium. Vertebral bone resorption was stimulated in a dose-dependent manner by parathyroid hormone (1–100 nM), 1,25 dihydroxyvitamin D₃ (0.0325–3.25 nM), and prostaglandin E₂ (3–3000 nM). Epidermal growth factor (300 ng/ml) produced a small stimulation of bone resorption which was not inhibited by indomethacin (0.5 μM). Likewise, indomethacin (0.5 μM) did not inhibit PTH-stimulated vertebral bone resorption. Calcitonin (6.6 nM) produced a 79% inhibition of bone resorption induced by PTH (10 nM), whereas estradiol (up to 3 μM) did not inhibit bone resorption. Our results demonstrate that estrogen does not have a direct effect on vertebral bone tissuein vitro. This new bone culture system is a sensitive assay for the direct effects of resorptive agents on vertebral bone.     

Low-dose parathyroid hormone and estrogen reverse alkaline phosphatase activity suppressed by dexamethasone in mouse osteoblastic cells

Glucocorticoid (GC)-induced osteoporosis (GIO) is frequently seen in patients with excessive GC. Numerous questions remain to be clarified about the pathogenesis and treatment of GIO, and the mechanism of GC-inhibited bone formation is not well known. Several studies suggest that parathyroid hormone (PTH) and hormone replacement therapy are effective for GIO. We therefore investigated whether PTH and estrogen would affect cell proliferation and alkaline phosphatase (ALP) activity inhibited by dexamethasone (Dex) in mouse osteoblastic cell-line MC3T3-E1 cells. Low-dose (10⁻¹¹ M) PTH as well as 10⁻⁸ M 17-β-estradiol (17β-E₂) significantly attenuated Dex-inhibited ALP activity, although 10⁻⁸ M PTH did not affect it. ICI 182780 (10⁻⁸ M) antagonized the effects of 17β-E₂ on Dex-suppressed ALP activity. Neutralizing anti-IGF-I antibody (3 μg/ml) blocked the reverse effects of 17β-E₂ on ALP activity suppressed by Dex. PTH (10⁻¹¹ M), but not 17β-E₂, significantly attenuated [³H]thymidine incorporation inhibited by Dex. On the other hand, PTH and estrogen did not affect the level of 11-β-hydrosteroid dehydrogenase type I mRNA increased by Dex. In conclusion, the present study demonstrated that low-dose PTH and estrogen reversed Dex-inhibited ALP activity in the mouse osteoblastic cell-line.     

Androgen receptors in osteoblast-like cell lines

Summary Although androgens exert major effects on bone remodeling, the mechanisms by which they exert their effects remain unclear. Recently, it has become apparent that receptors for sex steroids may be present in osteoblastic cells. We have examined several cell lines with osteoblastic phenotypes to determine if specific, high affinity androgen receptors are present. Two cell lines of human origin (Saos-2 and U2-OS) and one of rat origin (UMR-106.01) were studied. Androgen binding sites were present in all cell lines. Binding affinities were high (K_D=1.6−2.5×10⁻¹⁰ M), and similar to those in classical androgen target tissues (prostate, kidney). Concentrations were greater in the human cell lines (1277 and 1605 sites/cell) than in the rodent line (74 sites/cell). In the human cell lines androgen binding was also specific and typical of androgen receptors in other tissues. Specific estrogen binding was not present in the UMR-106.01 cells, and no estrogen receptors were detectable in the human cell lines using an enzyme-linked receptor immunoassay. Specific binding for progesterone was also absent in the UMR-106.01 cells, but progesterone receptors were detected immunologically in the Saos-2 (119 sites/cell) and U2-OS (118 sites/cell) lines. These findings indicate the presence of androgen receptors that are of similar character to those in classical androgen target tissues, and suggest that the study of these cell lines may be useful in the study of the regulation of androgen effects in osteoblasts.     

持续给予hPTH1-34对体外成骨细胞增殖与分化的影响

目的研究持续给予人甲状旁腺激素（human parathyroid hormone,hPTH）1-34对体外培养成骨细胞增殖与分化的干预作用。方法体外培养乳鼠成骨细胞,将细胞分为空白培养液对照组和10^-6、10^-7、10^-8mol·L^-1hPTH（1-34）3个不同剂量的给药组。每48h换1次液,换液时采用MTT法进行细胞增殖测定,同时检测细胞裂解液中的碱性磷酸酶（alkaline phosphatase,ALP）和骨钙素（bone gla protein,BGP）的水平,持续培养6d。结果持续给予hPTH1-34作用48h后,各给药组的MTT均明显高于对照组且各给药组间差异均有显著性（P〈0.05）,至第6天时各给药组则均明显低于对照组（P〈0.05）。ALP和BGP测定结果显示持续给予hPTH1-34作用48h后,10^-6mol·L^-1PTH给药组的BGP含量明显高于对照组（P〈0.01）,其余各组ALP及BGP含量与对照组比较差异虽无显著性,但均值高于对照组,至第6天各给药组均明显低于对照组（P〈0.05）。结论持续给予hPTH（1-34）对体外培养成骨细胞增殖与分化的影响与作用的时间和给药浓度密切相关。短期持续用药有促进作用,并且在10^-6-10^-8mol·L^-1浓度范围内呈剂量依赖性;长期持续用药抑制成骨细胞的增殖与分化,给药浓度越高抑制作用越强。     

Skeletal homeostasis in tissue-engineered bone.

Tissue-engineering strategies to stimulate bone regeneration may offer an alternative approach to conventional orthopaedic and maxillofacial surgical therapies. Over the last decade, significant advances have been accomplished in developing biomimetic matrices, growth factors, cell transplantation and gene delivery therapeutics to support new bone growth. However, it is not known if tissue-engineered bone recapitulates the biology of normal skeletal tissue in response to physiologic cues. Here, we report that bone formed by the differentiation of transplanted murine bone marrow stromal cells (BMSCs) responds to a systemically delivered calciotropic hormone. Ectopic ossicles in mice exposed to catabolic doses of parathyroid hormone (PTH) had increased numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts as compared to control mice. In contrast, treatment with anabolic doses of PTH promoted a marked increase in trabecular bone mass as analyzed by microcomputed tomography and histomorphometry. Our findings demonstrate that bone formed from transplanted BMSCs is responsive to normal physiologic signals, and can be augmented by the addition of a systemic anabolic agent. Because multiple and distinct ossicles can be generated in a single animal, this versatile system may be used to: (a) elucidate cellular/molecular mechanisms in bone regeneration; (b) study cell-to-cell interactions in the bone marrow microenvironment in health and disease; and (c) evaluate the efficacy of osteotropic agents that modulate bone turnover in vivo.     

Skeletal actions of intermittent parathyroid hormone: effects on bone remodelling and structure

Intermittent administration of parathyroid hormone peptides has anabolic skeletal effects and reduces fracture risk in postmenopausal women with osteoporosis but the cellular and structural mechanisms by which these effects are mediated have not been fully established. In cancellous and endocortical bone, there is evidence that both modelling and remodelling-based formation contribute to the increase in bone mass although the contribution of these at different time points in the response to PTH has not been established. Despite the large increase in spine bone mineral density, however, significant increases in iliac crest cancellous bone volume and trabecular thickness have not been consistently demonstrated, possibly reflecting site-specific differences in PTH-induced skeletal effects and/or the large sampling and measurement variance associated with assessment of iliac crest cancellous bone volume and structure. In iliac crest cortical bone, increased cortical thickness has been demonstrated, due at least in part to increased endosteal bone formation; there is also some evidence for increased formation on periosteal surfaces. At some sites an increase in cortical porosity may also occur and the overall effects on cortical bone strength, particularly at the hip, remain to be established. Studies in iliac crest bone indicate a trend towards a lower mineralisation density of bone matrix and increased heterogeneity of mineralisation, consistent with new bone formation. In addition, there is a reduction in mineral crystallinity and a shift towards more divalent collagen cross-links, indicating a change towards a younger bone profile.

The potential clinical implications of these effects on bone are currently unknown. The stimulatory effect of PTH peptides on bone formation may favour their use in low turnover bone disease and in states of advanced bone loss. Furthermore, if beneficial effects on cortical bone strength are confirmed, efficacy at non-vertebral sites might be superior to those observed with antiresorptive drugs. Better definition of the effects of intermittent PTH administration on cancellous and cortical bone remodelling and structure at different skeletal sites may inform these speculations and is an important area for future research.     

Acute Effects of Different Phosphorus Sources on Calcium and Bone Metabolism in Young Women: A Whole-Foods Approach

The recommended dietary phosphorus intake is exceeded in the typical Western diet. However, few studies have been conducted on the bioavailability and metabolic consequences of dietary phosphorus from different food sources. In this study, acute effects of dietary phosphorus from three different food sources and a phosphate supplement on calcium and bone metabolism were investigated. Sixteen healthy women aged 20–30 years were randomized to five controlled 24-hour study sessions, each subject serving as her own control. At the control session, calcium intake was ca. 250 mg and phosphorus intake ca. 500 mg. During the other four sessions, phosphorus intake was about 1,500 mg, 1,000 mg of which was obtained from meat, cheese, whole grains, or a phosphate supplement, respectively. The foods served were exactly the same during the phosphorus sessions and the control session; only phosphorus sources varied. Markers of calcium and bone metabolism were followed. Analysis of variance with repeated measures was used to compare the study sessions. Only the phosphate supplement increased serum parathyroid hormone (S-PTH) concentration compared with the control session (P = 0.031). Relative to the control session, meat increased markers of both bone formation (P = 0.045) and bone resorption (P = 0.049). Cheese decreased S-PTH (P = 0.0001) and bone resorption (P = 0.008). These data suggest that the metabolic response was different for different foods.     

地塞米松对不同月龄雌性大鼠成骨细胞雌激素受体表达的影响

目的观察体外培养的不同月龄雌性大鼠成骨细胞内雌激素受体水平的差异及地塞米松（1×10^-8mol/L）对不同月龄雌性大鼠成骨细胞雌激素受体表达的影响。方法采用酶消化法分别提取、纯化和培养新生（24h以内）、2、4、8、12月龄雌性Wistar大鼠成骨细胞,并且用Western-blot、ECL法检测体外培养成骨细胞雌激素受体的水平。结果提取的大鼠成骨细胞生长情况良好,碱性磷酸酶染色阳性,具有成骨细胞特性。Western-blot结果显示：雌性大鼠成骨细胞内雌激素受体水平在新生鼠和8月龄鼠较高,在2、4、12月龄鼠较低;地塞米松组成骨细胞雌激素受体表达水平在新生和2月龄低于对照组,在4、8、12月龄高于对照组。结论不同月龄雌性大鼠体外培养的成骨细胞雌激素受体的表达水平不同;1×10^-8mol/L地塞米松对体外培养的雌性大鼠成骨细胞雌激素受体水平有影响且与大鼠的月龄有关。     

Molecular mechanisms underlying the effects of sex steroids on bone and mineral metabolism

The multifarious functions of sex steroid receptors include a role as regulatory factors for bone and mineral metabolism in vivo. These functions are more complex than originally assumed. The finding of nuclear receptors in osseous tissue alludes to the existence of novel indirect and direct functions of bone tissue beyond skeletal support, hematopoiesis, and calcium homeostasis. Cell-specific gene targeting approaches are an extremely important technology for future studies that will need to be conducted to fully understand the molecular mechanisms underlying bone formation and metabolism. Y. Imai is a recipient of the 2007 JSBMR Research Encouragement Award.     

Skeletal Site-Dependent Expression of the Androgen Receptor in Human Osteoblastic Cell Populations

There are obvious sexual differences in adult skeletal morphology which for the most part are related to differences in size. Higher androgen serum levels in males exert potent osteoanabolic effects and therefore may contribute to this sexual dimorphism of the skeleton. The presence of androgen receptors (AR) in bone cells is a prerequisite for a direct osteoanabolic action of androgens. To investigate the possibility that, in addition to gender-related differences in androgen serum levels, there are also gender-related differences in the osteoblastic expression pattern of the androgen receptor, we examined AR mRNA expression, androgen binding sites, and mitogenic responses to the androgen dihydrotestosterone (DHT) in human osteoblastic cell (HOC) populations. HOCs were isolated from bone biopsy specimens derived from different skeletal sites of healthy adult males and females (2–69 years old). We found that male and female HOCs of all examined ages express similar AR mRNA levels and similar numbers of androgen binding sites. Using whole-cell-binding assays, we observed 3129–8417 androgen binding sites per femoral HOC with apparent KDs of 1.45–2.83 nM depending on the age of the investigated HOC population. Mandibular and cortical HOC of both sexes expressed higher AR mRNA levels, significantly more androgen binding sites per cell, and exhibited significantly greater mitogenic responses to DHT than iliac crest-derived and trabecular HOC of the same skeletal system and the same skeletal-site, respectively. In early adulthood, HOCs of both sexes appear to express somewhat higher AR mRNA levels and to possess more androgen binding sites than prepubertal and senescent HOC. Because sex hormone serum levels rise in puberty, we investigated the regulation of the AR mRNA expression by various steroids. We found that dexamethasone (dexa) and in some experiments also 17β-estradiol (E2) and 1,25-dihydroxyvitamin D3 (D3) increased AR mRNA levels and androgen binding in HOC cultures. A pretreatment with dexa, E2, and D3 significantly increased the mitogenic response of HOCs to DHT. We conclude that (1) higher androgen serum levels in males together with a higher AR expression at certain skeletal sites may contribute to the development of sex-related differences in skeletal morphology, (2) glucocorticoids induce AR gene expression in HOC cultures, and (3) glucocorticoids, E2, and D3 enhance the mitogenic action of DHT. Received: 3 June 1996 / Accepted: 30 April 1997     

持续性甲状旁腺素抑制成骨细胞分化 的实验研究

目的探讨持续性给与甲状旁腺素(PTH)对成骨细胞分化过程的抑制作用。方法培养MC3T3-E1成骨前体细胞,持续性给与PTH处理,碱性磷酸酶(ALP)染色方法,检测细胞内ALP的分泌;免疫荧光方法检测细胞内Osterix(Osx)蛋白的表达;real-time RT-PCT法和Westernblot方法检测细胞内成骨因子基因和蛋白的表达。结果 MC3T3-E1成骨前体细胞具有自发向成骨细胞方向分化的特征,与对照组相比,经PTH持续性处理的细胞ALP染色强度明显降低,Osx免疫荧光强度较弱,细胞内成骨因子基因和蛋白的表达亦显著低于对照组。结论持续性给与PTH具有抑制成骨细胞分化的作用。     

Control of intracellular pH in rat calvarial osteoblasts: Coexistence of both chloride-bicarbonate and sodium-hydrogen exchange

Summary Intracellular pH was monitored continuously in cultured rat calvarial osteoblasts using the pH-sensitive fluorescent dye bis carboxyethyl carboxyfluorescein (BCECF), loaded into the cells as its membrane permeant ester. Recovery from an intracellular acid load generated by exposure to NH₄Cl was unaffected by the anion exchange inhibitors 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disuphonic acid (SITS) and 4,4′-diisothiocyanatostilbene 2,2′-disuphonic acid (DIDS) (100 μM), but blocked by the sodium-hydrogen exchange inhibitor amiloride (1 mM) and dependent on external sodium, suggesting that recovery is brought about by a sodium-hydrogen exchanger in the plasma membrane. The cells do, however, possess a SITS-sensitive chloride-bicarbonate exchanger, because iso-osmotic replacement of chloride by gluconate leads to intracellular alkalinization, that is inhibited by SITS, but independent of external sodium. Parathyroid hormone brings about an intracellular acidification, which may be due to an inhibition of sodium-hydrogen exchange.     

闭经患者雌激素与雄激素对骨密度的影响

为了解原发闭经和继发闭经患者的雌雄激素水平与骨密度的关系,收集了１８～５１岁原发闭经患者９０例,继发闭经患者２６０例,并以相同年龄正常月经１８０例为对照组。分别测定其血清雌二醇和睾酮水平,测量皮质骨和松质骨骨密度。结果：原发闭经和继发闭经患者的雌激素均低于正常对照组。２１－羟化酶缺乏患者的雌激素水平稍低但雄激素远高于其它各类闭经患者和正常对照组。各类原发闭经和继发闭经患者的皮质骨和松质骨骨密度均低于同年龄正常对照组,唯独２１－羟化酶缺乏患者骨密度不但未降低反而升高３．１％。多囊性卵巢综合征患者的骨密度降低较少,它与２１－羟化酶缺乏二者均有高雄激素的临床特征。总之,原发闭经患者骨密度低于继发闭经患者,松质骨的骨密度较皮质骨更低。结果表明雌激素和雄激素缺乏均可影响骨密度,雌、雄激素缺乏越早,骨密度越低     

Characteristics of steroid hormone receptors in cultured MC3T3-E1 osteoblastic cells and effect of steroid hormones on cell proliferation

Summary We examined the binding characteristics of three kinds of steroid hormones—estrogen, androgen, and glucocorticoid—in cultured MC3T3-E1 mouse osteoblastic cells by whole-cell binding assay. The binding studies revealed the presence of a single class of high-affinity binding sites for [³H]17-estradiol, [³H]mibolerone (a synthetic androgen), and [³H]triamcinolone acetonide (a synthetic glucocorticoid). The numbers of binding sites for these steroid hormones were found to be 4534±819, 14312±1884, and 24898±655 sites/cell; and the Kd values were 8.57±0.62 x 10^–10M, 1.12±0.19 x 10^–9 M, and 6.08±1.24 x ^–10M, respectively. We also examined the effects of steroid hormones on the proliferation of MC3T3-E1 cells. 17-estradiol significantly stimulated the proliferation of the cells (130–150% of control). Dihydrotestosterone also significantly stimulated the proliferation of the cells (115% of control); the effect was, however, much less potent than that of 17-estradiol, although the number of binding sites was approximately three times more than that of 17 estradiol. Triamcinolone acetonide and dexamethasone had no effect on cell proliferation. These results suggest that estrogen and androgen act directly on osteoblastic cells through a receptor-mediated mechanism, and that androgen is much less potent than estrogen in stimulating the proliferation of MC3T3-E1 osteoblastic cells.     

Bone endothelial cells as estrogen targets

Summary In the present study, we investigated the effects of estrogens on bone endothelial cell metabolism and the presence of estrogen binding sites in the same cells. For these studies, we have used a continuous cell line of clonal bovine bone endothelial cells for evidence of a direct response to estrogensin vitro. Receptor analysis to intact viable cells was steroid specific and saturable, with an apparent dissociation constant of 17.2 nM and a B_max of 3.2 × 10⁴ sites/cell. Northern blot analysis revealed a 6.5-kilobase mRNA that hybridized with a cDNA to human estrogen receptor. The 6.5-kilobase size is in close agreement with the reported size of the human estrogen receptor mRNA.In vitro estrogen responses of bone endothelial cells included a stimulation of cell proliferation as well as an inhibition of parathyroid hormone responsiveness. These findings clearly demonstrate the presence of functional estrogen receptors in bone endothelial cellsin vitro, suggesting a role of estrogens in bone angiogenesis and in the entire process of bone remodeling.     

不同剂量的甲状旁腺素对成骨细胞分化的不同作用的实验研究

目的探讨不同剂量的甲状旁腺素(PTH)对成骨细胞分化过程的影响。方法培养MC3T3-E1成骨前体细胞,给与不同浓度的PTH处理细胞,real-time PCR方法,检测细胞内成骨因子基因mRNA的表达;碱性磷酸酶(ALP)染色方法,检测细胞内ALP的分泌;Alizarin Red染色方法,检测细胞内成骨钙化作用。结果 1 nmol/L浓度的PTH和10 nmol/L浓度的PTH都具有明显的促进成骨细胞分化的作用,以10 nmol/L浓度的PTH的作用更强。100 nmol/L较高浓度的PTH没有明显促进成骨细胞分化的作用。结论不同剂量的PTH对成骨细胞的分化作用,具有不同的影响。