在酵母中异源表达人破骨细胞钠氢转运蛋白

目的 在酿酒酵母中异源表达人破骨细胞分化成熟潜在因子钠氢转运蛋白2(HsNHA2),鉴定其作为盐离子载体转运Na+的功能,并对其在细胞中的表达进行初步定位分析.方法 采用高保真PCR试剂盒扩增出HsNHA2;构建酵母表达载体,通过电穿孔的方式转入到酿酒酵母菌株中;Western印迹检测其在酵母菌株的表达;通过NaCl选择压力观察菌株在高盐环境中的生长表型,并用根皮素抑制HsNHA2观察其抗盐功能;最后用细胞原位荧光染色确定HsNHA2在细胞中的表达位置.结果 Western印迹证实HsNHA2在酵母菌株中正常表达;生长曲线显示HsNHA2可使菌株在高盐环境中生长,根皮素抑制HsNHA2表达后菌株不能在0.2mol/L NaCl培养基中生长;免疫荧光染色初步证明HsNHA2表达在细胞膜上.结论 HsNHA2作为盐离子载体在酵母细胞中主要表达在细胞膜上行使转运Na+的功能,以维持细胞正常生长.     

Intracellular signal transduction pathways: good therapeutic targets for joint destruction in rheumatoid arthritis

Preventing joint destruction is one of the most challenging issues in treating patients with rheumatoid arthritis (RA), and I propose that intracellular signaling pathways in osteoclasts and synovial fibroblastic cells (SFCs) can be good therapeutic targets. Osteoclasts are primarily involved in the bone destruction in RA joints, and SFCs support osteoclast differentiation and activation by producing various proinflammatory cytokines including receptor activator of NF-B ligand (RANKL), the osteoclast differentiation factor belonging to the tumor necrosis factor- superfamily. Suppressing c-Src pathways by adenovirus vector-mediated C-terminal Src family kinase (Csk) gene or Ras/extracellular-regulating kinase (ERK) pathways by introducing dominant negative Ras (Ras^DN) adenovirus reduced osteoclastic bone resorption as well as the abnormal proliferation and interleukin-6 production of SFCs, and the local injection of these viruses ameliorated the joint destruction in adjuvant arthritis rats. Moreover, chondrogenic differentiation of SFCs could be induced by stimulating activin receptor-like kinase 3 pathways.     

The role of osteoblast cells in the pathogenesis of unicameral bone cysts

Purpose

The pathogenesis of unicameral bone cysts (UBCs) remains largely unknown. Osteoclasts have been implicated, but the role of osteoblastic cells has, to date, not been explored. This study investigated the pathophysiology of UBCs by examining the interactions between the cyst fluid and human bone marrow stromal cells (hBMSCs) and the effect of the fluid on osteogenesis.

Methods

Fluid was aspirated from two UBCs and analysed for protein, electrolyte and cytokine levels. Graded concentrations of the fluid were used as culture media for hBMSCs to determine the effects of the fluid on hBMSC proliferation and osteogenic differentiation. The fibrocellular lining was analysed histologically and by electron microscopy.

Results

Alkaline phosphatase (ALP) staining of hBMSCs that were cultured in cyst fluid demonstrated increased cell proliferation and osteogenic differentiation compared to basal media controls. Biochemical analysis of these hBMSCs compared to basal controls confirmed a marked increase in DNA content (as a marker of proliferation) and ALP activity (as a marker of osteogenic differentiation) which was highly significant (p < 0.001). Osteoclasts were demonstrated in abundance in the cyst lining. The cyst fluid cytokine profile revealed levels of the pro-osteoclast cytokines IL-6, MIP-1α and MCP-1 that were 19×, 31× and 35× greater than those in reference serum.

Conclusions

Cyst fluid promoted osteoblastic growth and differentiation. Despite appearing paradoxical that the cyst fluid promoted osteogenesis, osteoblastic cells are required for osteoclastogenesis through RANKL signalling. Three key cytokines in this pathway (IL-6, MIP-1α, MCP-1) were highly elevated in cyst fluid. These findings may hold the key to the pathogenesis of UBCs, with implications for treatment methods.     

Differences in osteoclast formation between proximal and distal tibial osteoporosis in rats with adjuvant arthritis: inhibitory effects of bisphosphonates on osteoclasts

Abstract

Patients with rheumatoid arthritis commonly suffer both systemic and periarticular osteoporosis. Bisphosphonates (BPs) are inhibitors of bone resorption, and several derivatives have been developed for treatment of enhanced bone resorption. We aimed to characterize osteoclast formation in two different sites, the proximal tibial and distal tibial areas, in rats with adjuvant arthritis, and to investigate the impact of amino or non-amino types of bisphosphonate. Adjuvant arthritis was initiated in rats while administering daily injections of either etidronate, a non-amino BP, or alendronate, an amino BP, for 3 weeks. On the day following the last injection, bone mineral density (BMD) was measured in the proximal tibia to assess systemic osteoporosis and in the distal tibia for periarticular osteoporosis using dual-energy X-ray absorptiometry. Subsequently, bone marrow cells from either end of the tibia were collected and incubated for 7 days before staining and counting tartrate-resistant acid phosphatase positive cells. In the rats with adjuvant arthritis, BMD of either end of the tibia was lower than in normal rats. Although etidronate prevented bone mineral loss at both ends, distal loss was significantly less than proximal. In contrast, alendronate significantly inhibited mineral loss primarily in the proximal area. Large osteoclasts, defined as having five or more nuclei, formed preferentially in the proximal tibia, while small osteoclasts with fewer than four nuclei were found mainly distally. The suppressive effect of alendronate was greater on the large osteoclasts, while etidronate had a greater effect on the small osteoclasts. These results show that the size and multinuclearity of osteoclasts and the number of osteoclasts formed are different in the distal and proximal areas of the tibia, and that alendronate and etidronate may suppress different types of osteoclasts as discriminated by the number of nuclei.     

The Role of Osteoblast Density and Endogenous Interleukin-6 Production in Osteoclast Formation From the Hemopoietic Stem Cell Line FDCP-MIX C2GM in Coculture With Primary Osteoblasts

Osteoclast formation from the hemopoietic stem cell line FDCP-mix C₂GM was shown to be strongly dependent on osteoblast density. In cocultures of C₂GM cells with fetal mouse osteoblasts seeded at high density (i.e., 2.5 × 10⁴ cells/cm²), we found a significantly lower osteoclast formation compared with cocultures with osteoblasts seeded at low density (i.e., 1 × 10⁴ cells/cm²). The differentiation state of osteoblasts in high-density cultures resembled more than that of osteoblasts in low-density cultures, the differentiation state of mature osteoblasts, since the cells in the former cultures showed higher alkaline phosphatase (APase) activity than the cells in the latter cultures, and nodules were formed in high-density cultures but not in low-density cultures. Endogenous interleukin-6 (IL-6) production was found to be significantly lower in high-density cultures, which may partly explain the impaired osteoclast formation in high-density cocultures. Addition of IL-6 to the high-density cocultures indeed restored osteoclast formation. There appeared to be no overt difference in IL-6 receptor mRNA expression between high-density and low-density cultures. In conclusion, this paper suggests that mature, highly differentiated osteoblasts are not directly involved in osteoclastogenesis. In contrast, osteoblast-like cells lacking mature osteoblast markers induce osteoclast formation. Whether these low-density osteoblast-like cells represent an immature differentiation state or the lining cell phenotype is unclear. Received: 26 June 1997 / Accepted: 14 November 1997     

双膦酸盐对骨形成蛋白诱导骨骨吸收抑制作用的研究

目的研究双膦酸盐(bisphosphonate YM 175)对骨形成蛋白(bone morphogenetic protein．简称BMP)诱导骨骨吸收的抑制作用。方法 42只大白鼠背部植入BMP,诱导出异位骨后,将大白鼠分成2组,即投药组和对照组。投药组在BMP植入后第3w至第7w,双膦酸盐(YM 175)每周投药3次,剂量1(μg／kg．d)。对照组按同样的方式给予等量的生理盐水。在BMP植入第3w、 4w、7w和10w,将BMP诱导骨取出,采用TRAP和cathepsin K染色方法来观察双膦酸盐对破骨细胞的作用。结果 BMP诱导骨3w时(即未投双膦酸盐药前),在BMP植入体周边形成编织骨 (woven bone),大量破骨细胞出现在新生骨组织表面。在4 w时,双膦酸盐投药组和对照组,新生骨组织均向BMP植入体内生长,但双膦酸盐投药组的破骨细胞较对照组有减少。在10 w时,双膦酸盐投药组和对照组均可观察到骨细胞变小并且有规律地排列的板层状骨(lamellar bone)的特征。而双膦酸盐投药组,破骨细胞死亡,与对照组比较,破骨细胞数目明显减少。结论双膦酸盐对破骨细胞性骨吸收有明显的抑制作用。     

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.     

Osteoclasts and Biomaterials

Abstract There is a growing market of biomaterials for orthopedic applications. As soon as these materials are surgically introduced into the constantly remodeling bone of the patient, they start to interact with the local cells: osteoblasts and osteoclasts. At the first glance, the bone building osteoblasts seem to be the more important cells for osseointegration of implants. However, it is mainly the bone resorbing action of osteclasts that determines the longevity of the implant. In this paper, we give a short overview over the current understanding of osteoclast biology; we review the interaction between biomaterials, biomaterial particles and osteoclasts, and the effects of treatment with antiosteoclastic agents like bisphosphonates on biomaterial implant healing.     

破骨细胞的分化及其调节研究进展

破骨细胞（Osteoclast OC）来源于单核/巨噬细胞谱系,其数量和活性的不足或增加分别会导致骨硬化症和骨质疏松及其他溶骨性疾病。为了更好的理解这些疾病的发病机理和制定相关治疗方案,我们就必须揭示破骨细胞的分化调节机制。笔者将就破骨细胞在分化过程中的信号通路及其调节因素以及一些潜在可行的治疗骨量丢失相关疾病的方法予以综述。     

1,25(OH)2维生素D3诱导小鼠混合培养细胞白细胞介素1α mRNA的表达

目的本研究旨在探讨破骨细胞形成过程中１，２５（ＯＨ）２维生素Ｄ３［１，２５（ＯＨ）２Ｄ３］和白细胞介素１α（ＩＬ１α）两种生物因子间的相互作用关系，以期进一步了解正畸牙齿移动过程中牙周组织改建的生物学机理。方法ＲＴＰＣＲ将ＩＬ１αｍＲＮＡ表达信号放大后，采用Ｓｏｕｔｈｅｒｎ法检测混合培养细胞在１，２５（ＯＨ）２Ｄ３作用下，ＩＬ１αｍＲＮＡ的表达。结果发现ＩＬ１αｍＲＮＡ的表达强度与１，２５（ＯＨ）２Ｄ３浓度呈正相关关系，在时间顺序上ＩＬαｍＲＮＡ在第２天表达最强，第４天、第６天相对减弱。结论证明ＩＬ１参与１，２５（ＯＨ）２Ｄ３对破骨细胞生成的诱导作用。推测前４天促进破骨细胞前体增殖，后２天促进其分化。