云南乌骨绵羊乌质性状与TYR基因多态性的相关分析

乌骨乌肉是乌骨绵羊的主要乌质性状。本文测定了乌骨绵羊、兰坪本地羊和罗姆尼羊血液TYR活性并分析了乌骨绵羊与乌骨鸡组织器官黑色素结构,结果表明：乌骨绵羊TYR活性显著高于兰坪本地绵羊和罗姆尼羊（P<0.05）;乌骨绵羊黑色素与乌骨鸡黑色素的IR光谱基本一致,黑色物质主要是真黑色素。首次克隆了绵羊TYR基因第一外显子长667 bp序列,并检测了乌骨绵羊和非乌骨绵羊TYR基因多态性。结果发现,检测的乌骨绵羊TYR基因第一外显子有两个变异位点,分别位于第64和154号编码氨基酸上,但都属于同义突变。通过对64号位置设计酶切位点检测TYR基因多态性,结果发现,该突变与乌质性状有关基因紧密连锁,TYR基因上可能存在乌质性状相关功能突变位点。另外,TYR基因多态性与毛色的表型相关极显著（P<0.01）,该基因可能也是毛色功能基因。     

Functions of RANKL/RANK/OPG in bone modeling and remodeling

The discovery of the RANKL/RANK/OPG system in the mid 1990s for the regulation of bone resorption has led to major advances in our understanding of how bone modeling and remodeling are regulated. It had been known for many years before this discovery that osteoblastic stromal cells regulated osteoclast formation, but it had not been anticipated that they would do this through expression of members of the TNF superfamily: receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), or that these cytokines and signaling through receptor activator of NF-κB (RANK) would have extensive functions beyond regulation of bone remodeling. RANKL/RANK signaling regulates osteoclast formation, activation and survival in normal bone modeling and remodeling and in a variety of pathologic conditions characterized by increased bone turnover. OPG protects bone from excessive resorption by binding to RANKL and preventing it from binding to RANK. Thus, the relative concentration of RANKL and OPG in bone is a major determinant of bone mass and strength. Here, we review our current understanding of the role of the RANKL/RANK/OPG system in bone modeling and remodeling.     

OPG/RANKL/RANK系统与骨破坏性疾病

近年来发现的OPG/RANKL/RANK系统在破骨细胞生成中起着至关重要的作用,是骨骼生理研究领域的重大进展。成骨细胞、骨髓基质细胞、激活的T淋巴细胞表达RANKL,与破骨细胞前体细胞或成熟破骨细胞表面上的RANK结合后,促进破骨细胞的分化及骨吸收活性。成骨细胞及骨髓基质细胞分泌表达OPG可与RANKL竞争性结合,从而阻断RANKL与RANK之间的相互作用。体内多种激素或因子通过影响骨髓微环境内的OPG/RANKL比率来调节骨代谢。此外,乳腺上皮细胞表达有RANK,孕期在性激素的诱导下可表达RANKL,OPG/RANKL/RANK系统在孕期乳腺发育以及母体向胎儿的钙转运过程中发挥重要作用。阻断RANKL/RANK通路有望给骨质疏松、类风湿关节炎及癌症骨转移等骨破坏性疾病的治疗开辟新的途径。进一步研究应了解OPG/RANKL/RANK系统与其它信号传导途径的关系,重视骨骼、免疫及内分泌系统之间的相互作用。目前,开发与OPG功能相似或促进其表达的合成药物有可能成为具有良好经济效益和社会效益的产业。     

Heritable disorders of the RANKL/OPG/RANK signaling pathway

Figure 7 summarizes the heritable disorders identified to date that directly involve the RANKL/OPG/RANK signaling pathway in humans. Activating mutations in TNFRSF11A encoding RANK and deactivating mutations in TNFRSF11B encoding OPG cause systemic bone disease (FEO, PDB2, ESH and JPD) featuring accelerated bone turnover, low bone mass, deafness early in life, and loss of dentition by enhancing signaling. No human disease has been identified involving defects in the TNFSF11 gene encoding RANKL. Despite genetic bases for these autosomal dominant and recessive conditions involving bone cell receptors, focal expansile osteolytic lesions are common and can occur perhaps from further local activation of osteoclast-mediated bone resorption following trauma. These disorders resemble PDB which can be inherited as an autosomal dominant trait with focal osteolytic disease, sometimes with deafness and tooth loss, and increasingly associated with mutations, but in other genes.     

In vivo anti-osteoporotic activity of isotaxiresinol, a lignan from wood of Taxus yunnanensis

Isotaxiresinol, the main lignan isolated from the water extract of wood of Taxus yunnanensis, was investigated for its effect on bone loss, on serum biochemical markers for bone remodeling and on uterine tissue, using ovariectomized (OVX) rats as the model of postmenopausal osteoporosis. After oral administration of isotaxiresinol (50 and 100mg/kg/d) for 6 weeks, bone mineral content (BMC) and bone mineral density (BMD) in total and cortical bones were increased as compared to those of OVX control rats, and decreases of three bone strength indexes induced by OVX surgery were prevented. Serum biochemical markers for bone remodeling revealed that isotaxiresinol slightly increased bone formation and significantly inhibited bone resorption without side effect on uterine tissue. These results suggest that isotaxiresinol may be useful for treatment of postmenopausal osteoporosis, especially for prevention of bone fracture induced by estrogen deficiency.     

The OPG/RANKL/RANK system in metabolic bone diseases

The OPG/RANKL/RANK cytokine system is essential for osteoclast biology. Various studies suggest that human metabolic bone diseases are related to alterations of this system. Here we summarize OPG/RANKL/RANK abnormalities in different forms of osteoporoses and hyperparathyroidism. Skeletal estrogen agonists (including 17beta-estradiol, raloxifene, and genistein) induce osteoblastic OPG production through estrogen receptor-alpha activation in vitro, while immune cells appear to over-express RANKL in estrogen deficiency in vivo. Of note, OPG administration can prevent bone loss associated with estrogen deficiency as observed in both animal models and a small clinical study. Glucocorticoids and immunosuppressants concurrently up-regulate RANKL and suppress OPG in osteoblastic cells in vitro, and glucocorticoids are among the most powerful drugs to suppress OPG serum levels in vivo. As for mechanisms of immobilization-induced bone loss, it appears that mechanical strain inhibits RANKL production through the ERK 1/2 MAP kinase pathway and up-regulates OPG production in vitro. Hence, lack of mechanical strainduring immobilization may favor an enhanced RANKL-to-OPG ratio leading to increased bone loss. As for hyperparathyroidism, chronic PTH exposure concurrently enhances RANKL production and suppresses OPG secretion through activation of osteoblastic protein kinase A in vitro which would favour increased osteoclastic activity. In sum, the capacity for OPG to antagonize the increases in bone loss seen in many rodent models of metabolic bone disease implicates RANKL/OPG imbalances as the likely etiology and supports the potential role for a RANKL antagonist as a therapeutic intervention in these settings.     

Multiple myeloma/hypercalcemia

Multiple myeloma, a cancer of plasma cells, is associated with excessive tumor-induced, osteoclast-mediated bone destruction. Hypercalcemia remains the most frequent metabolic complication of myeloma in patients, and excessive osteolysis plays a major contributory role in its pathogenesis. The clinical presentation of hypercalcemia in patients varies depending on the level of ionized calcium; it can be life threatening, as in the case of hypercalcemic crisis, requiring immediate medical treatment to prevent death. During the past few years there have been exciting developments in our understanding of the pathogenesis of myeloma bone disease; in particular, key mediators of the osteoclastic bone resorption in myeloma have been identified, including receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage inflammatory protein-1alpha. There is also increasing evidence that Dickkopf 1, which has been shown to be over-expressed in myeloma patients, is also a potent stimulator of osteoclast formation and activity. Importantly, the available data suggest that RANKL is the final common mediator of osteoclastic bone resorption, irrespective of the upstream initiator molecule. This brief review presents an overview of the roles played by these mediators in inducing osteolysis in myeloma bone disease, and it discusses targeting RANKL as a potential new treatment strategy in myeloma bone disease and myeloma-associated hypercalcemia.     

The effect of imidazole and imidazole-analogues on bone resorption in vitro: A suggested role for thromboxane A2

The effects of imidazole, 1-methyl-imidazole and benzimidazole on bone metabolism $\text{in vitro}$ were investigated. The relative potencies of these compounds with respect to the inhibition of bone resorption was found to be comparable to their relative effectiveness as inhibitors of platelet microsome thromboxane synthetase activity. Since studies by others have shown that thromboxanes are produced by resorbing bone $\text{in vitro}$, these results suggest that the inhibition of bone resorption by imidazole is related to the inhibition of thromboxane A₂ formation. This could imply that thromboxane A₂ is an additional arachidonic acid oxidation product that is of importance in the regulation of bone metabolism.     

交联I型胶原复合生物玻璃在兔股骨远端缺损中的成骨作用

摘要 目的：评价交联度不同的I型胶原复合生物玻璃后作为人工骨移植物在兔股骨髁部骨缺损中的修复作用,以研究一种成骨性能优秀,降解速度令人满意,且具有可塑性,便于术中使用的新型人工骨移植材料。方法：本研究设置实验组及对照组,实验组为交联度70%的高交联I型胶原复合生物玻璃以及交联度为45%的低交联I型胶原复合生物玻璃。对照组为普通未交联I型胶原复合生物玻璃。于9只新西兰大白兔双下肢股骨髁部制备动物骨缺损模型,将随机分组后的三种骨移植物分别植入股骨髁部骨缺损模型中。术后6周取材行组织学分析研究,比较3种骨移植物在骨缺损中的新骨生成率。结果：组织学分析结果显示,高交联组的新骨生成率为5.23 0.87%,其成骨性能显著低于低交联组13.23 1.13%以及未交联组的12.63 0.92%（P<0.05）。而低交联组的新骨生成率与未交联组之间无统计学差异（P>0.05）。结论：交联度为45%的低交联I型胶原复合生物玻璃具有更好的成骨能力,作为骨移植材料在临床应用中具有更广阔的发展前景。     

T cell regulation of interferon-alpha/beta (IFN-alpha/beta) production by alloantigen-stimulated bone marrow cells

We have previously reported that mouse bone marrow cells produce high levels of interferon-alpha/beta (IFN-alpha/beta) after 5 to 6 days of in vitro culture with irradiated allogenic spleen cells. The current study was initiated to determine whether or not T cells are important for alloantigen-induced IFN-alpha/beta production by mouse bone marrow cells. Bone marrow cells and spleen cells were obtained from C57BL/6 mice. These cells were treated with different monoclonal antisera and complement, and then were cultured 5 to 6 days with irradiated DBA spleen cells. The results from these experiments indicated that optimal IFN-alpha/beta production by alloantigen-stimulated bone marrow cells required Lyt-1+2+ T cells. In addition, when bone marrow cells obtained from nu/nu B10 mice were cultured with alloantigen, only low levels of IFN were produced when compared with IFN production by bone marrow cells obtained from normal littermate B10 mice. The addition of nylon wool-enriched splenic T cells to cultures containing bone marrow cells and alloantigen resulted in an augmentation of IFN-alpha/beta production by three-fold to fivefold. Furthermore, bone marrow cells obtained from alloantigen-immunized mice produced much higher levels of IFN-alpha/beta and in a shorter period of time (2 to 3 days) when compared with bone marrow cells obtained from control or non-immunized mice. Cyclosporin A (CsA) has been shown to inhibit predominantly T cell-dependent responses. The effect of CsA on IFN production by alloantigen-stimulated bone marrow and spleen cells was investigated. The addition of CsA at concentrations as low as 0.1 micrograms/ml inhibited not only IFN-gamma production by alloantigen-stimulated spleen cells, but also IFN-alpha/beta production by alloantigen-stimulated bone marrow cells. In contrast, IFN-alpha/beta production by Newcastle disease virus-infected spleen cells, bone marrow cells, or L cells was not inhibited by the addition of CsA (1 microgram/ml). Thus, the ability of bone marrow cells to produce high levels of IFN-alpha/beta after in vitro culture with alloantigen is dependent upon T cells resident in the bone marrow. IFN-alpha/beta production by alloantigen-stimulated bone marrow cells may play a major role in the pathogenesis associated with graft-vs-host disease and in T cell regulation of hematopoiesis.     

Enhancement of bone formation ex vivo and in vivo by a helioxanthin-derivative

To effectively treat serious bone defects using bone-regenerative medicine, a small chemical compound that potently induces bone formation must be developed. We previously reported on the osteogenic effect of 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin-derivative, in vitro. Here, we report on TH’s osteogenic effects ex vivo and in vivo. TH-induced new bone formation in both calvarial and metatarsal organ cultures. A novel monitoring system of osteoblastic differentiation using MC3T3-E1 cells revealed that TH was released from α-TCP bone cement and this release continued for more than one month. Lastly, the implantation of the α-TCP carrier containing TH into defects in mouse skull resulted in increased new bone areas within the defects after 4 weeks. A TH-containing scaffold may help establish a more efficient bone regeneration system.     

Comparison of rat mandible bone characteristics in f344 substrains, f344/du and f344/n.

The characteristics of the mandible bone were compared through DXA methods between two major substrains of F344 rats, F344/DuCrlCrlj and F344/NSlc at around 60 days of age. Since these two substrains are clearly different in survival and mandible morphology, some genetic differences are supposed to exist. In contrast to a previous microsatellite analysis, clear and significant differences were detected in the body and mandible weights, the mandible bone mineral contents (BMC), bone area (AREA), bone mineral density (BMD) and bone mineral ratio (BMR), between F344/DuCrlCrlj and F344/NSlc, with the mandible molar teeth intact in the bone. Thus, care is needed in the experimental use of these substrains, as results may differ between them. The newly proposed parameter, BMR, may especially contribute to the comparison of bone characteristics among species.     

OPG/RANK/RANKL系统与骨质疏松研究最新进展

骨质疏松是严重威胁中老年人健康的骨科常见病,OPG/RANK/RANKL是参与调节骨重建的最重要的分子系统之一,与骨疾病相关的骨质疏松有密切联系,并已成为药物设计的新靶点.因此,对该系统的深入研究将为骨生理、病理机制阐明及骨疾病防治带来积极影响.     

Role of Slit/Robo Signaling pathway in Bone Metabolism

Slit/Robo signals were initially found to play an essential role in nerve development as axonal guidance molecules. In recent years, with in-depth study, the role of Slit/Robo in other life activities, such as tumor development, angiogenesis, cell migration, and bone homeostasis, has gradually been revealed. Bone is an organ with an active metabolism. Bone resorption and bone formation are closely related through precise spatiotemporal coordination. There is much evidence that slit, as a new bone coupling factor, can regulate bone formation and resorption. For example, Slit3 can promote bone formation and inhibit bone resorption through Robo receptors, which has excellent therapeutic potential in metabolic bone diseases. Although the conclusions of some studies are contradictory, they all affirm the vital role of Slit/Robo signaling in regulating bone metabolism. This paper reviews the research progress of Slit/Robo signaling in bone metabolism, briefly discusses the contradictions in the existing research, and puts forward the research direction of Slit/Robo in the field of bone metabolism in the future.     

Prostaglandin A2 and 35S uptake in connective tissue

Rats deficient in essential fatty acids (EFA) incorporated lesser amounts of radioactive sulfate into lung, kidney, spleen, heart, costal cartlidge, long bone and skull bone than did normal control animals. Administration of prostaglandin A₂ stimulated ³⁵S uptake by lung, kidney and aorta while ³⁵S levels in costal cartilage, tibial cap and long bone were strikingly reduced. Comments are presented suggesting that this metabolic mechanism may explain, in part, cartilage and bone resorption in areas of inflammation, such as arthritis, both rheumatoid arthritis and osteoarthritis.     

Bone cancer pain and the role of RANKL/OPG

Cancer-induced bone diseases are common and can have a devastating impact at the end of life. One of the most difficult sequelae of cancer is metastases to the skeleton, an event that results in bone destruction and bone cancer pain. Bone cancer pain is usually progressive as the disease advances, and is particularly difficult to treat. Recently, experimental models of bone cancer pain have been developed and have provided seminal insight in understanding the pathophysiology of bone cancer pain. Animal models of bone cancer provided the finding that bone destruction (osteolysis) is associated with pain, and it has been determined that cancer-induced osteolysis is mediated by osteoclasts. Having established that RANK ligand contributed to cancer-induced osteoclastogenesis, it was determined that disruption of the RANKL-RANK axis with OPG inhibited tumor-induced osteoclastogenesis and decreased bone cancer pain.     

Antiosteoclastic bone resorption activity of osteoprotegerin via enhanced AKT/mTOR/ULK1-mediated autophagic pathway

Autophagy plays a critical role in the maintenance of bone homeostasis. Osteoprotegerin (OPG) is an inhibitor of osteoclast-mediated bone resorption. However, whether autophagy is involved in the antiosteoclastogenic effects of OPG remains unclear. The present study aimed to investigate the potential mechanism of autophagy during OPG-induced bone resorption via inhibition of osteoclasts differentiated from bone marrow-derived macrophages in BALB/c mice. The results showed that after treatment with receptor activator of nuclear factor-κΒ ligand and macrophage colony-stimulating factor for 3 days, TRAP⁺ osteoclasts formed, representing the resting state of autophagy. These osteoclasts were treated with OPG and underwent autophagy, as demonstrated by LC3-II accumulation, acidic vesicular organelle formation, and the presence of autophagosomes. The levels of autophagy-related proteins, LC3-II increased and P62 decreased at 3 hr in OPG-treated osteoclasts. The viability, differentiation, and bone resorption activity of osteoclasts declined after OPG treatment. Treatment with OPG and chloroquine, an autophagy inhibitor, attenuated OPG-induced inhibition of osteoclastic bone resorption, whereas rapamycin (RAP), an autophagy inducer, enhanced OPG-induced inhibition of differentiation, survival, and bone resorption activity of osteoclasts. Furthermore, OPG reduced the amount of phosphorylated(p) protein kinase B (AKT) and pmTOR and increased the level of pULK, in a dose-dependant manner. LY294002, a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT pathway inhibitor, attenuated the decline in pAKT, but enhanced the decline in pmTOR and the increase in pULK1 following OPG treatment. RAP enhanced the OPG-induced increase in pULK1. The PI3K inhibitor 3-methyladenine partly blocked OPG-induced autophagy. Thus, the results revealed that OPG inhibits osteoclast bone resorption by inducing autophagy via the AKT/mTOR/ULK1 signaling pathway.