Effects of androgens on subpopulations of the human osteosarcoma cell line SaOS2

Previously, we showed that androgens stimulate murine and human osteoblast-like cell proliferation and differentiation by mechanisms involving increased responses to mitogenic growth factors (GF) and increased GF production. To explain this dual action of androgens on primary osteoblastic cell populations we advanced the hypothesis that androgens exert differential effects on osteoblastic subpopulations. We subcloned a human osteosarcoma cell line (SaOS2) into subpopulations expressing high (HAS) and low (LAS) levels of alkaline phosphatase (ALP). The obtained subclones differed significantly in their ALP production and expressed a high and low ALP phenotype, respectively, for the entire experimental period. Dihydrotestosterone (DHT) increased specific ALP activity and type-I procollagen peptide secretion in both HAS and LAS. DHT pretreatment enhanced the mitogenic action of basic fibroblast growth factor (bFGF) and insulinlike growth factor 2 (IGF2) only in HAS. The enhanced mitogenic effect of IGF2 in HAS after DHT pretreatment was associated with increased IGF2-receptor mRNA levels. Therefore, we conclude that androgens exert their osteoanabolic action (1) by stimulating differentiated functions of osteoblastic cells with a high and a low ALP phenotype, and (2) via increased growth factor receptor expression and thereby enhancing mitogenic growth factor responses only in HAS. This paper is dedicated to the occasion of Prof. Dr. R. Ziegler's 60th birthday     

Collagenase and Tissue Plasminogen Activator Production in Developing Rat Calvariae: Normal Progression Despite Fetal Exposure to Microgravity

Exposure to zero gravity has been shown to cause a decrease in bone formation. This implicates osteoblasts as the gravity-sensing cell in bone. Osteoblasts also are known to produce neutral proteinases, including collagenase and tissue plasminogen activator (tPA), which are thought to be important in bone development and remodeling. The present study investigated the effects of zero gravity on development of calvariae and their expression of collagenase and tPA. After in utero exposure to zero gravity for 9 days on the NASA STS-70 space shuttle mission, the calvariae of rat pups were examined by immunohistochemistry for the presence and location of these two proteinases. The ages of the pups were from gestational day 20 (G20) to postnatal (PN) day 35. Both collagenase and tPA were found to be present at all ages examined, with the greatest amount of both proteinases present in the PN14 rats. At later ages, high amounts were maintained for tPA but collagenase decreased substantially between ages PN21 to PN35. The location of collagenase was found to be associated with bone-lining cells, osteoblasts, osteocytes, and in the matrix along cement lines. In contrast, tPA was associated with endothelial cells lining the blood vessels entering bone. The presence and developmental expression of these two proteinases appeared to be unaffected by the exposure to zero gravity. The calvarial thickness of the pups was also examined; again the exposure to zero gravity showed little to no effect on the growth of the calvariae. Notably, from G20 to PN14, calvarial thickness increased dramatically, reaching a plateau after this age. It was apparent that elevated collagenase expression correlated with rapid bone growth in the period from G20 to PN14. To conclude, collagenase and tPA are present during the development of rat calvariae. Despite being produced by the same cell in vitro, i.e., the osteoblast, they are located in distinctly different places in bone in vivo. Their presence, developmental expression, and quantity do not seem to be affected by a brief exposure to zero gravity in utero. Received: 6 May 1997 / Accepted: 27 January 1998     

Fatty Acids and Bone

Nutritional status is an important determinant of skeletal health. Increased fat mass favorably influences bone mineral density and reduces fracture risk. The mechanisms by which adiposity influences skeletal health include mechanical skeletal loading, the effects of adipocyte and pancreatic β-cell-derived hormones that act on bone, and neuroendocrine outputs from the hypothalamus that respond to peripheral nutritional signals. A growing body of evidence, including the recognition that specific fatty acid receptors are expressed in skeletal tissue, also suggests that fatty acids affect skeletal health. These effects include indirect actions of dietary and circulating fatty acids, mediated by hormonal signals derived from the intestine and pancreas, and direct effects of some fatty acid species on bone cells. Emerging evidence suggests that marrow adipocyte-derived fatty acids might affect skeletal health via paracrine mechanisms. The existing evidence suggests that there is potential for both positive and negative effects of fatty acids on the skeleton, such that the net effect may be context-specific. Careful laboratory and clinical investigation is required to increase our understanding of a potentially important area of skeletal health.     

孔雀绿比色法同步测定大鼠成骨细胞膜Ca~(2+)-ATP酶和Na~+、K~+-ATP酶活性

为探索同步测定大鼠成骨细胞膜Ｃａ２＋－ＡＴＰ酶和Ｎａ＋、Ｋ＋－ＡＴＰ酶活性的方法。采用孔雀绿比色法测定磷以反映酶活性。结果显示：Ｃａ２＋浓度以及成骨细胞传代培养的代数是影响酶活性的重要因素。Ｃａ２＋有助于提高ＡＴＰａｓｅ活性。Ｃａ２＋浓度为４０μｍｏｌ／Ｌ时，ＡＴＰａｓｅ活性较高。细胞传代越多，膜Ｃａ２＋－ＡＴＰａｓｅ活性越低。成骨细胞膜Ｃａ２＋－ＡＴＰａｓｅ活性低于Ｎａ＋、Ｋ＋－ＡＴＰａｓｅ。结论：孔雀绿比色法同步测定大鼠成骨细胞膜Ｃａ２＋－ＡＴＰ酶和Ｎａ＋、Ｋ＋－ＡＴＰ酶活性，方法简便、灵敏、可靠。     

成骨细胞株与强韧化纳米人工骨支架联合培养实验研究

目的 :观察成骨细胞株 ( 3T3 E1)在新型纳米氧化锆强韧化高孔隙率人工骨支架 (下文简称人工骨支架 )材料上生长情况。方法 :成骨细胞株 ( 3T3 -E1)与人工骨支架联合培养 ,通过光镜观察和细胞计数的方法了解成骨细胞与支架结合能力 ,扫描电镜观察细胞生长的情况。结果 :新型强韧化纳米人工骨材料与建株成骨细胞有良好的结合能力 ,成骨细胞株 ( 3T3 E1)在人工骨材料上生长良好。结论 :纳米骨支架是较理想的骨组织工程支架材料 ,成骨细胞复合支架用于骨缺损的修复 ,具有广阔的临床应用前景。     

核结合因子a1的调控机制

Cbfa1是骨发育过程中调节骨髓基质干细胞向成骨细胞分化和成熟的重要转录因子。Cbfa1的表达水平异常与骨骼系统疾病有关。体内体外实验证实多种通路(如Wnt/LRP5/-catenin,BMP/Smads,1,25-(OH)2-vitaminD3/VDR/VDRE途径)和调节蛋白(Msx2,Dlx5,Twists)在Cbfa1基因表达、活性和随后的骨形成过程中起关键作用。这些发现对调控成骨细胞分化和治疗骨质疏松以及其他伴有骨量改变的疾病治疗提供了新的思路,这些疾病有可能用控制Cbfa1表达来进行治疗。     

GSB中药血清对rMSCs定向诱导分化为成骨细胞的影响

目的观察GSB中药血清对大鼠MSCs定向诱导分化成骨的影响。方法将培养的rMSCs分为对照组（C组）[含10%胎牛血清,100U/ml青霉素和100μg/ml链霉素的完全培养液]、诱导组（O组）[完全培养液,加诱导培养液（0.1μmol/L地塞米松、10mmol/Lβ-甘油磷酸钠及50μg/ml维生素C）,加20%空白血清]和含药血清诱导组（G组）[完全培养液加诱导培养液诱导培养液中加入20%含药血清）。显微镜下观察细胞形态变化,生化法测定上清中Ca^2＋浓度,Gomori改良钙钴法进行ALP染色,Von Kossa法进行矿化结节染色,RT-PCR法测定Ⅰ型胶原（COLL1）和脂蛋白脂酶（LPL）的表达,放免法测定上清中骨钙素含量。结果O组和G组在加入诱导培养液3-4天后梭形细胞比例减少,细胞变形成多角形、乃至方形,部分区域细胞成多层生长;改良Gomori钙钻法碱性磷酸酶染色阳性细胞,Von Kassa染色发现钙盐沉积;培养9d后细胞Ca^2＋浓度开始升高,并随时间持续显著增长,且G组明显高于O组。自第15d后,O组和G组细胞ALP活性随时间延长而明显增高,且G组显著高于O组;培养的第14d,G组骨钙素分泌量明显高于O组。成骨诱导剂作用MSCs12d后,COLLⅠ mRNA表达阳性,LPL mRNA表达阴性,且G组比O组COLLⅠ mRNA表达更强。结论补肾填精中药GSB促进骨钙素分泌、增强ALP活性、促钙盐沉积,具有增强成骨诱导剂诱导rMSCs向成骨细胞分化,并促进成骨细胞成熟作用。     

Effects of transforming growth factor type β on expression of cytoskeletal proteins in endosteal mouse osteoblastic cells

Transforming growth factor β (TGFβ) has been shown to influence the growth and differentiation of many cell types in vitro. We have examined the effects of TGFβ on cell morphology and cytoskeletal organization in relation to parameters of cell proliferation and differentiation in endosteal osteoblastic cells isolated from mouse caudal vertebrae. Treatment of mouse osteoblastic cells cultured in serum free medium for 24 hours with TGFβ (1.5–30 ng/mL) slightly (− 23%) inhibited alkaline phosphatase activity. In parallel, TGFβ (0.5–30 ng/mL, 24 hours) greatly increased cell replication as evaluated by [³H]-thymidine incorporation into DNA (157% to 325% of controls). At a median dose (1.5 ng/mL) that affected both alkaline phosphatase and DNA synthesis (235% of controls) TGFβ induced rapid (six hours) cell respreading of quiescent mouse osteoblastic cells. This effect was associated with increased polymerization of actin, actinin, and tubulins, as evaluated by both biochemical and immunofluorescence methods. In addition, TGFβ (1.5 ng/mL) increased the de novo biosynthesis of actin, actinin, vimentin, and tubulins, as determined by [³⁵S] methionine labeling and fractionation of cytoskeletal proteins using two-dimensional gel electrophoresis. These effects were rapid and transient, as they occurred at six hours and were reversed after 24 hours of TGFβ exposure. The results indicate that the stimulatory effect of TGFβ on DNA synthesis in endosteal mouse osteoblastic cells is associated with a transient increase in cell spreading associated with enhanced polymerization and synthesis of cytoskeletal proteins.     

Relationship of bone morphogenetic protein expression during osteoblast differentiation to wild type p53

We have previously shown p53 to have a specific role in osteoblast differentiation by its ability to regulate expression of certain bone specific proteins. In this study, we show mineralized matrix formation in vivo to be directly related to the presence of wild type p53 in osteoblastic osteosarcoma cells. In order to further understand the importance of p53 in differentiation, we investigated the relationship between p53 and Bone Morphogenetic Proteins (BMPs) (BMP 1, 2, 3A, 3B (GDF-10), 4, 5, 6, 7, 8A and 8B) during osteoblast differentiation. The expression of several BMPs were tested using RNase Protection Assay in differentiating ROS17/2.8 osteoblastic osteosarcoma cells. The expression of BMPs 1, 2, 3a, 3b and 7 showed time dependent modulation during in vitro differentiation. In order to determine if p53 has a role in this process, we used a murine osteosarcoma cell line stably expressing a temperature sensitive p53. Cells were exposed to ascorbic acid and glycerophosphates to hasten in vitro osteoblast differentiation and maintained either at 32 or 37 degrees C for expression of the wild type or mutant p53 phenotype. The expression of BMP-2, BMP-4 and BMP-7 were modulated in a p53 dependent fashion. We were able to confirm the p53 dependency of BMP-2 independently by RT-PCR. While BMP-2 expression was evident in the presence of both wild type and mutant p53, regulated expression was seen only in cells expressing wild type p53. Transient over expression of wild type p53 did not result in the same BMP-2 response as stable expression showing that the presence of p53 may be important for an orderly development of osteoblast differentiation rather than a direct effect on gene expression. The functional relationship between p53 and these bone specific markers is discussed.     

人成骨细胞瘦素受体放射配基结合分析研究

骨质疏松是以骨量减少、骨组织微结构被破坏为特征的系统性骨病。研究表明 ,无论是否是负重骨 ,血清雌激素水平经校正后 ,体脂和骨量直接相关 ,因此肥胖被认为是骨量的保护因子[1～ 3 ] 。瘦素(leptin)是肥胖基因 (ob)的蛋白质表达产物 ,主要由脂肪细胞分泌 [4 ] 。已有的研究表明 ,瘦素不仅是降低食欲、调节能耗和体重的重要体脂激素 ,而且还与机体多种代谢功能密切相关。有研究提出 ,瘦素可能是骨形成的合成因子 ,介导肥胖对骨量的影响 [5,6]。本研究拟通过培养成人成骨细胞 (osteoblast,OB) ,采用受体放射分析方法探讨 OB瘦素受体蛋白 …