瑞舒伐他汀对体外培养人成骨细胞增殖及LRPS、Runx2基因表达的影响

目的 观察不同浓度的瑞舒伐他汀对体外培养的人成骨细胞增殖及LRPS,Runx2基因表达的影响,探讨瑞舒伐他汀影响骨代谢可能的分子机制。方法 (1)成骨细胞的培养与鉴定;(2)给予不同浓度(0,10-6 , 10-7 ,10-8,10-9 mol/L)的瑞舒伐他汀刺激体外培养的人成骨细胞,24 h后采用CCK-8比色法检测细胞的增殖能力,用荧光定量PCR法检测LRPS , Runx2的相对表达水平。结果 瑞舒伐他汀可明显促进细胞增殖(P<0.05),并促进成骨细胞LRPS , Runx2基因的表达(P<0.05),10-7mol/L增殖最明显、基因的表达量最高。结论 瑞舒伐他汀可促进成骨细胞的增殖,可能与LRPS , Runx2基因表达增加有关。     

Simultaneous measurement of bone formation and resorptionin vivo

Summary Bone resorption and formation in terms of milligram of calcium deposited into and released from whole humerus of 15–23 weeks old male rats were assessed, via linear least-squares regression analysis of change in calcium content and³H-tetracycline kinetics of the bone. The rate of resorption was 0.376 mg calcium per day, while formation was 0.758 mg calcium per day. The data indicate that the computation results in a simultaneous and explicit measurement of bone resorption and formation at the organ level.     

24,25(OH)2D3, bone formation,and bone resorption in vitamin D-deficient,azotemic rats

Summary Bone formation, mineralization, and resorption were measured in vitamin D-deficient, azotemic rats given two different dosages of 24,25(OH)₂D₃ daily and in vehicle-treated controls (C). The intraperitoneal administration of 65 pmol over a 10 day period corrected the hypocalcemia observed in C, whereas 130 pmol produced mild hypercalcemia. Both dosages reduced osteoid width, osteoid area, and mineralization front width form control values. The rates of bone and matrix formation were unaffected by treatment. In C, matrix formation exceeded bone formation and resulted in osteoid accumulation; both dosages of 24,25(OH)₂D₃ reversed this relationship such that bone formation exceeded matrix formation in each treatment group. The rates of osteoid maturation and initial mineralization increased during repletion with 24,25(OH)₂D₃ at both dosage levels. However, the serum calcium concentration was correlated with both osteoid maturation rate (r=0.68,P<0.01) and initial mineralization rate (r=0.63,P<0.01) when all three experimental groups were considered. Bone resorption was unchanged from control values during treatment with 24,25(OH)₂D₃. The results suggest that 24,25(OH)₂D₃ promotes the maturation and mineralization of osteoid, and that this metabolite differs in its effects on bone formation and resorption. It is not clear, however, that the changes in bone dynamics observed are independent of the calcemic response induced by metabolite repletion under the conditions of this experiment.     

Effects of mechanical strain on proliferation and differentiation of bone marrow stromal cell line ST2

Differentiation of mesenchymal stromal cells into osteoblasts is regulated by many factors including growth factors, cytokines, and hormones. Mechanical stress has been considered to be an important factor in bone modeling and remodeling. However, biological responses of stromal cells to mechanical stimuli are still unknown. To show the correlation between magnitude of mechanical strain and differentiation of stromal cells into osteoblasts, we investigated the proliferation and the expression of osteoblast-related genes in stromal cell line ST2 that is in the process of osteoblastic differentiation by treatment with ascorbic acid and -glycerophosphate, under 0.8%–15% elongation using the Flexercell Strain system. The expression of osteoblast-related genes was analyzed by real-time quantitative polymerase chain reaction (PCR). Cell proliferation significantly increased at 5%, 10%, and 15% elongation compared to that of unloaded controls. Alkaline phosphatase (ALPase) activity significantly increased at 0.8% and 5% elongation but decreased at 10% and 15% elongation. At 1h and 6h, mRNA level of Cbfa1/Runx2 increased at lower magnitudes of strain (0.8% and 5% elongation) but decreased at higher magnitude of strain (15% elongation). At 24 and 48h, Cbfa1/Runx2 and osteocalcin mRNAs decreased at 5%, 10%, and 15% elongation, whereas cell proliferation and expression of type I collagen mRNA increased at the same elongation. These results indicate that mechanical strain stimulates osteoblastic differentiation of stromal cells at low magnitudes of strain.     

Plasma proteins in human cortical bone: Enrichment ofα 2 HS-glycoprotein,α 1 acid-glycoprotein,and IgE

Summary Human cortical bones were extracted with EDTA, and the residue after EDTA extraction was digested with bacterial collagenase. Ten plasma proteins were identified and quantitated in the EDTA extracts. Three of them—IgE, IgD, andα ₁acid-glycoprotein—had not previously been described in bone or dentine. Five plasma proteins identified in collagenase digests are albumin, IgG, IgA, IgE, andα ₁acid-glycoprotein. IgE,α ₁acid-glycoprotein, andα ₂HS-glycoprotein were found to be concentrated in the bone more than other plasma proteins by factors between 11 and 525. The identification of plasma proteins was facilitated by the addition of polyethylene glycol in agarose gel. The presence of plasma proteins both in EDTA extracts and in collagenase digests suggests their structural role in bone.     

CYP1A2, CYP2D6, GSTM1, GSTP1, and GSTT1 gene polymorphisms in patients with bladder cancer in a Turkish population

Genetic differences in the metabolism of xenobiotics have recently been suggested as modifiers of individual susceptibility to bladder cancer (BC). The objective of this study was to investigate the relationship between bladder tumor and variants of cytochrome p450 1A2 (CYP1A2) 734 C → A, cytochrome p450 2D6 (CYP2D6) 1934 G → A, glutathione S-transferase M1, (GSTM1 null), glutathione S-transferase T1 (GSTT1 null), and glutathione S-transferase P1 (GSTP1) I105 V. We investigated the distribution of these polymorphisms in 135 BC patients and in 128 age and sex-matched cancer-free controls. The polymorphisms were analyzed using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay and the multiplex PCR method. Genotype and allele frequencies and their associations with BC risk, demographic factors, smoking status, and tumor stage were investigated. The prevalence of GSTT1 null genotype in cases was 23%, compared with 7% in the control group (OR = 3.94, 95% CI = 1.70–9.38, P = 0.001). There was no association between the studied polymorphisms of CYP1A2, CYP2D6, GSTM1, and GSTP1 genes and BC. There was an association between smoking status and BC. These data seem to indicate that GSTT1 gene polymorphism may be associated with BC in the Turkish population studied. Further studies will be needed to clarify the role of such variation in determining susceptibility to BC.     

补肾益髓中药复方对去卵巢骨质疏松症大鼠骨组织Runx2 mRNA及蛋白表达的影响

目的观察去卵巢骨质疏松症大鼠骨组织Runx2 mRNA及蛋白表达,探讨绝经后骨质疏松症(Postmenopausal Osteoporosis,PMOP)的发病机制以及补肾益髓中药复方的疗效机理。方法去卵巢复制骨质疏松症大鼠模型,实验设正常组、模型组、假手术组、补肾益髓中药复方组、钙尔奇D组、骨疏康组。术后7d开始灌胃给药12周。应用XR-36型双能X线骨密度仪测定股骨骨密度,应用OLYMPUS BX51显微镜观察股骨头石蜡切片HE染色显微形态结构,实时定量RT-PCR及Western Blot检测骨组织Runx2 mRNA及蛋白表达。结果 (1)与正常组、假手术组比较,模型组股骨骨密度明显降低(P0.001、P0.05),骨组织Runx2 mRNA及蛋白表达明显降低(P0.001)。(2)与模型组比较,补肾益髓中药复方组、骨疏康组股骨骨密度明显升高(P0.05),补肾益髓中药复方组(P0.001)、钙尔奇D组(P0.05)、骨疏康组(P0.01)骨组织Runx2 mRNA及蛋白表达明显上调。(3)与钙尔奇D组、骨疏康组比较,补肾益髓中药复方组骨组织Runx2 mRNA(P0.01、P0.05)及蛋白(P0.001)表达均明显升高。(4)股骨头石蜡切片HE染色显微形态结构:与正常组、假手术组比较,模型组骨小梁形态结构完整性差,有些部位破坏、断裂,余留骨重建空间较多;各给药组骨小梁形态结构均改善,结构较紧密,余留骨重建空间减少,其中以补肾益髓中药复方组改善最明显。结论骨组织Runx2 mRNA及蛋白表达降低可能是PMOP的发病机制之一;补肾益髓中药复方可能通过上调骨组织Runx2 mRNA及蛋白表达有效防治PMOP,其作用优于钙尔奇D、骨疏康。     

Alcohol consumption and the risk of breast cancer among BRCA1 and BRCA2 mutation carriers

Alcohol consumption increases the risk of breast cancer among women in the general population, but its effect on women who carry a BRCA gene mutation is unclear. We conducted a case-control study of 1925 matched pairs of predominantly premenopausal women who carry a BRCA1 or a BRCA2 mutation. Information on current alcohol consumption was obtained from a questionnaire administered during the course of genetic counselling or at the time of enrolment. A modest inverse association between breast cancer and reported current alcohol consumption was observed among women with a BRCA1 mutation (OR = 0.82, 95% CI 0.70–0.96), but not among women with a BRCA2 mutation (OR = 1.00; 95% CI 0.71–1.41). Compared to non-drinkers, exclusive consumption of wine was associated with a significant reduction in the risk of breast cancer among BRCA1 carriers (p-trend = 0.01). Alcohol consumption does not appear to increase breast cancer risk in women carrying a BRCA gene mutation.     

Association of PLXNA2 polymorphisms with vertebral fracture risk and bone mineral density in postmenopausal Korean population

Introduction Plexin A2 (PLXNA2) is a receptor that recognizes secreted or membrane-bound semaphorin 3A, which is implicated in neural regulation of bone metabolism.Materials and Methods In the present study, we identified 48 genetic polymorphisms in PLXNA2 by resequencing, and 10 single nucleotide polymorphisms (SNPs) were selected for further investigation into their potential involvement in osteoporosis in a postmenopausal population (n=560).Results Two SNPs, +14G>A (Gln5Arg) and +183429C>T (Tyr1621Tyr), and Block1-ht2 were associated with risk of vertebral fracture (p=0.01–0.05), and three SNPs, +799G>A (Ala267Thr), +135391G>A, and +190531G>C, were associated with bone mineral density at various femur sites (p=0.003–0.03). Particularly, the minor allele of +14G>A was associated with a protective effect on vertebral fracture and higher lumbar bone mineral density, suggesting that +14G>A may be a useful marker for osteoporosis and its related fracture.Conclusion These results provide, for the first time, evidence supporting the association of PLXNA2 with osteoporosis in postmenopausal women. Electronic Supplementary Material Supplementary material is available for this article at     

Effects of thionapthene 2-carboxylic acid and related compounds on bone resorption in organ culture

Summary We have compared the effects of thiophene 2-carboxylic acid (TCA) and a number of sulfur-and nitrogen-containing analogs for their ability to inhibit bone resorption in organ cultures of fetal rat long bones. Four compounds,—thionapthene-2-carboxylic acid (TNCA), dibenzo-thiophene-4-carboxylic acid, indole-2-carboxylic acid and carbazole-1-carboxylic acid—caused a doserelated inhibition of PTH-stimulated bone resorption, although TCA was ineffective in this system. TNCA at 3×10⁻⁴ M or 10⁻⁴ M was the most potent inhibitor of PTH-stimulated bone resorption and was selected for further study. TNCA also inhibited stimulation of resorption by prostaglandin E₂ and 1,25-dihydroxyvitamin D. Unlike calcitonin, the effect of TNCA was persistent and did not show escape. Moreover, TNCA could inhibit resorption in bones that had previously escaped from calcitonin. TNCA did not appear to be a nonspecific toxin, since it did not decrease incorporation of [³H] thymidine or [³H]proline into fetal rat long bones. The fact that resorption in unstimulated cultures was only decreased when the control rates were high also argues against nonspecific toxicity. Moreover, this suggests that TNCA will be most effective under conditions of accelerated bone resorption when an inhibiting effect is most desirable. heterocyclic sulfur-containing compound, thiophene-2 carboxylic acid (TCA) in the rat. Subsequent studies showed that this compound could inhibit bone resorption in organ cultures of neonatal mouse calvaria [3]. We have compared TCA with a number of analogs for their ability to inhibit PTH stimulated bone resorption in organ culture. TCA itself was found to be relatively ineffective, whereas several of the analogs were inhibitory at concentrations of 3×10⁻⁴ or 10⁻⁵ M. One of the most potent compounds, thionapthene 2-carboxylic acid (TNCA) was selected for further study. TNCA was a potent inhibitor not only of PTH stimulated bone resorption, but of resorption stimulated by prostaglandin E₂ (PGE₂) and 1,25-dihydroxyvitamin D (1,25-(OH)₂D₃). It was less effective in inhibiting resorption in control unstimulated cultures. TNCA did not appear to act as a nonspecific toxin in that it did not decrease [³H]-thymidine or [³H]-proline incorporation into bones. The inhibitory effect of TCA was persistent, unlike that of calcitonin, which shows escape after initial inhibition. Moreover, TNCA had a powerful inhibitory effect when it was added to bones that had previously escaped from CT. A preliminary report of this work was presented at the sixth annual meeting of the American Society for Bone and Mineral Research, Hartford, CT. 1984 [1]     

Effect of recombinant human granulocyte colony-stimulating factor (rh G-CSF) on rat bone: Inhibition of bone formation at the endosteal surface of vertebra and tibia

The effect of recombinant human granulocyte colony-stimulating factor (rh G-CSF) on bone was evaluated by histomorphometry using Sprague-Dawley rats. rh G-CSF was injected at doses of 0, 50, 150, and 450 μg/kg for 6 weeks.In vivo double fluorochrome labeling was performed before sacrifice. No significant change in body weight was observed. Bone mineral density (BMD) of lumbar vertebrae and femora was significantly decreased in G-CSF-treated groups. In the lumbar vertebra, osteoid surface, osteoid thickness, trabecular thickness, and labeled surface in G-CSF-treated groups were also significantly lower. In addition, osteoclast number and osteoclast surface were significantly higher in the G-CSF-treated groups. The endocortical surface at the mid-tibia showed lower labeled surface and mineral apposition rate in G-CSF-treated groups, without significant changes at the periosteal surface. Furthermore, numerous granulocytes fully occupied the bone marrow area. We conclude that proliferating granulocytes in the bone marrow may inhibit bone-forming cells from contacting the bone surface, resulting in reduction of bone formation;and increased osteoclastic bone resorption induced by G-CSF treatment contributed to the reduction of BMD.     

Adriamycin inhibits PTH-mediated but not PGE2-mediated stimulation of cyclic AMP formation in isolated bone cells

Summary We have examined the effect of adriamycin, an anthracycline antibiotic which modifies plasma membrane functions, on the cyclic AMP response to PTH and PGE₂ in isolated osteoblastlike cells. Adriamycin blunted the increment in bone cell cyclic AMP caused by exposure to PTH. This effect appeared rapidly (within 3 min after bone cells were exposed to adriamycin) and disappeared soon after exposure of adriamycin-treated cells to adriamycin-free incubation medium. Inhibition was evident over the entire time course of PTH action, at low as well as high PTH concentrations, and was one-half maximal at 31 μM adriamycin. It could not be attributed to alterations in cyclic AMP exodus, degradation or interference with the cyclic AMP assay, nor to impaired cell viability. Adriamycin also reduced the stimulatory effect of PTH on adenylate cyclase activity in a crude plasma membrane preparation. By contrast, adriamycin failed to modify the effects of PGE₂ on cyclic AMP generation in intact bone cells, and on adenylate cyclase activity in broken cells. Moreover, concentrations of adriamycin that blunted the effect of PTH on adenylate cyclase activity did not inhibit the stimulatory effects of sodium fluoride or of GppNHp. These results suggest that adriamycin selectively alters the interaction between PTH and its receptor or impairs the transmission of information from hormone-receptor complex to adenylate cyclase (or both), perhaps by binding to specific lipid domains in the plasma membrane. Structural analogues of adriamycin, which vary in their lipophilic properties, also varied in their capacity to perturb the cyclic AMP response. One such analogue in fact inhibited the response to PGE₂, and several appeared to augment the PGE₂ effect. These substances may well be useful in probing the membrane properties required for selectivity in hormone action. Supported by NIH grant AM 19855-07     

Establishment of a rapid bone resorption in vitro assay using previiously frozen mouse unfractionated bone cells pretreated with parathyroid hormone

We established a useful assay system for evaluating osteoclast-mediated bone resorption based on the use of unfractionated bone cells obtained from 10- to 11-day-old mice. When cells from 10 to 11 mice were treated for 7 days with rat parathyroid hormone (rPTH, 10⁻⁸ M), a total of 4 to 5 × 10⁷ cells could be obtained from the culture by treatment with 0.05% trypsin and 0.02% EDTA in PBS. These harvested cells contained about 20% tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells. When the harvested cells were cultured on dentine slices without rPTH, after 1 day, they formed TRAP-positive multinucleate cells that were active in bone rsorption. Eel calcitonin (eCT) decreased the number of pits in a dose-dependent manner, and its half maximal inhibition dose (ID₅₀) was 1.08 × 10⁻¹¹ M.Even after having been frozen in liquid nitrogen for 5 months, upon thawing, these cells were capable of forming pits; and this pit formation was inhibited by eCT. Since no appropriate osteoclaslic cell line for evaluating bone resorption is available at present, this system can provide a useful, practical means for assaying osteoclastic bone-resorbing activity.     

Anabolic effect of prostaglandin E2 on cortical bone of aged male rats comes mainly from modeling-dependent bone gain

In this study, prostaglandin E₂ (3 mg/kg per day) was administered to 20-month-old male Wistar rats for 10 and 30 days. Histomorphometric analyses were performed on double-fluorescent-labeled undecalcified tibial shaft sections. Thirty days of prostaglandin E₂ (PGE₂) administration increased bone formation rate/total bone surface from undetectable levels to 0.6 μm/day at the periosteal surface and from 0.5 to 2.1 μm/day at the endocortical surface. Endocortical osteoid surface area increased from 2% to 67% at day 10 and decreased to 6% at day 30; woven and lamellar bone formation started at day 0, but was most obvious at day 30, resulting in a 12% increase of total bone mass. The red to yellow marrow ratio was 0.2 in pretreatment controls, and increased to 1.6 by day 10 and 2.4 by day 30 with PGE₂ administration. Intracortical cavity number and area increased after 10 days of PGE₂ treatment, but with forming osteon number and area far exceeding those of resorption cavities at day 30. Endocortical modeling surface/endocortical surface was only 1.5%, and remodeling was 11.1% in pretreatment controls. PGE₂ treatment increased modeling to 24.5% in the 10 day group and 93.7% in the 30 day group, whereas remodeling remained unchanged at 10 days, and decreased to 6.2% at 30 days. Osteoprogenitor cells and osteoblasts could not be detected in pretreatment controls, but increased by day 10, and returned almost to control levels by 30 days. Our data indicate that PGE₂ induced periosteal and endocortical bone formation mainly by modeling-dependent bone gain, accompanied by increases in intracortical remodeling and red bone marrow, and a transient increase in the osteoprogenitor cells adjacent to the endocortical surface. These findings suggest that 20-month-old male Wistar rats were very responsive to the anabolic action of PGE₂ in the tibial shaft, a site consisting mainly of cortical bone and yellow marrow.     

Effects of long-term daily administration of prostaglandin-E2 on maintaining elevated proximal tibial metaphyseal cancellous bone mass in male rats

Summary The effects of long-term prostaglandin E₂ (PGE₂) on cancellous bone in proximal tibial metaphysis were studied in 7-month-old male Sprague-Dawley rats given daily subcutaneous injections of 0, 1, 3, and 6 mg PGE₂/kg/day and sacrificed after 60, 120, and 180 days. Histomorphometric analyses were performed on double fluorescent-labeled undecalcified bone specimens. After 60 days of treatment, PGE₂ produced diffusely labeled trabecular bone area, increased trabecular bone area, eroded and labeled trabecular perimeter, mineral apposition rate, and bone formation rate at all dose levels when compared with age-matched controls. In rats given PGE₂ for longer time periods (120 and 180 days), trabecular bone area, diffusely labeled trabecular bone area, labeled perimeter, mineral apposition, and bone formation rates were sustained at the elevated levels achieved earlier at 60-day treatment. The eroded perimeter continued to increase until 120 days, then plateau. The observation that continuous systemic PGE₂ administration to adult male rats elevated metaphyseal cancellous bone mass to 3.5-fold of the control level within 60 days and maintained it for another 120 days indicates that the powerful skeletal anabolic effects of PGE₂ can be sustained with continuous administration.     

Bone metabolic disorders caused by small bowel resection,and the influence of 1α-hydroxyvitamin D3 on the rats

Metabolic bone disorders caused by resection of the distal three quarters of the small bowel and the effects of 1α-hydroxyvitamin D₃ (1α(OH)D₃) on the rats were investigated biochemically and histomorphologically. 1α(OH)D₃ was administered orally to the rats for 13 weeks. A group of “ sham operation” animals and the 75%-distal-small-bowel-resected control animals received only the vehicle. Bone changes developed 13 weeks after the partial bowel resection. The disorder was characterized by reductions in ash content of the femur and by the disappearance of the trabecular bone in tibial metaphysis. Decreased Ca absorption from the intestines was demonstrated by a radiotracer technique, and biochemical studies showed significant decreases in serum Ca and 1,25(OH)₂D concentrations in the bowel-resected rats. These findings suggest that 75% distal small bowel resection impairs intestinal absorption of calcium and results in a negative calcium balance, which may contribute to the development of bone metabolic disorder in rats. This disorder was demonstrated as high bone turnover via urinalysis and by a radiotracer technique. Administration of 1α(OH)D₃ increased dose-dependent Ca absorption from the intestine and the serum Ca, normalized the bone turnover rate and prevented the development of bone metabolic disorder.