Juvenile Paget's disease, familial expansile osteolysis and other genetic osteolytic disorders

Several rare inherited osteolytic disorders have been described that show phenotypic overlap with Paget's disease of bone (PDB). Familial expansile osteolysis, early-onset familial PDB and expansile skeletal hyperphosphatasia are related disorders caused by mutations affecting the TNFRSF11A gene, which encodes the receptor activator of NFkappaB (RANK). The mutations result in failure of normal processing of RANK and osteoclast activation. Inactivating mutations in the TNFRSF11B gene, which encodes osteoprotegerin, cause idiopathic hyperphosphatasia which is a severe disorder that shares phenotypic similarities with PDB. The syndrome of hereditary inclusion body myopathy, PDB and frontotemporal dementia is caused by mutations in the gene encoding for valosin-containing protein which is involved in regulating degradation of ubiquitinated proteins. Anecdotal reports indicate that osteoclast inhibitors such as bisphosphonates are effective for suppressing bone turnover and improving symptoms in these disorders, although the long-term effects on clinical outcomes are unclear.     

BMP-2和OPG在腭横缝牵引中的表达

目的：了解腭横缝牵张成骨中骨形成蛋白2（BMP-2）和护骨素（OPG）的表达，探讨持续牵引力对骨改建的调控过程。方法：以杂种犬16只为实验动物，实验组在腭横缝及额上颌缝安置内置式镍钛合金牵引器。空白对照组不处理。术后10、20、30、40d切取腭横缝组织，行组织学观察。结果：实验组上颌骨成功前移，BMP-2、OPG阳性染色主要定位于成骨细胞中。结论：缝牵引可以顺利扩张生长期上颌骨，其成骨方式是膜内成骨；BMP-2可以诱导和激发成骨细胞活性，OPG在骨吸收过程中起重要调控作用。     

淫羊藿苷对人牙周膜细胞增殖和骨保护素mRNA表达的影响

目的：研究淫羊藿苷对人牙周膜细胞（periodontal ligament cells，PDLCs）增殖和骨保护素（osteoprotegerin，OPG）mRNA表达的影响。方法：体外培养人PDLCs，用MTT法检测不同浓度的淫羊藿苷（0．001、0．01、0、1μg／mL）、不同时间（24、48、72、96h）作用下人PDLCs的增殖水平；RT-PCR检测OPG mRNA的表达。结果：淫羊藿苷（0、001-0．1μg／mL）对人PDLCs增殖和OPG mRNA表达具有促进作用（P〈0．01），0．01μg／mL浓度作用最明显。结论：淫羊藿苷能够促进人PDLCs增殖和OPG mRNA表达。     

骨保护素/核因子-κB受体活化因子/核因子κB-受体活化因子配体信号分子调控牙萌出的研究进展

牙萌出是牙胚、牙槽骨以及多种类细胞系及多条通路信号分子相互作用共同调控的、复杂有序的生理过程。牙齿萌出需要在牙胚的方形成萌出通道,穿过牙槽骨和口腔黏膜到达功能位置。这一过程主要由破骨细胞直接执行,而骨保护素/核因子-κB受体活化因子/核因子κB-受体活化因子配体信号分子(OPG/RANK/RANKL)信号分子则可通过调节破骨细胞的分化与成熟来调控牙槽骨的改建,以保证牙萌出时方牙槽骨能被正常吸收。牙萌出的具体调控机制尚不明确,该文就OPG/RANK/RANKL信号分子调控牙萌出的研究现状作一综述。     

人甲状旁腺激素(1-34)突变蛋白的设计与活性验证

通过对NCBI数据库中不同物种同源序列进行比对protein-protein BLAST,并利用计算机软件DiscoveryStudio3.1进行分子对接和分子动力学模拟,对天然人甲状旁腺激素(1 34)的3个氨基酸R25K26K27进行Q25E26L27置换,并对突变体的生物活性进行了评价。结果显示:突变后PTH(1 34)-(RKK-QEL)和突变前PTH(1 34)多肽主链叠合后均方根偏差RMSD值为2.509 3,说明两者主链结构构象差异不大;PTH(1 34)-(RKK-QEL)与受体蛋白PTH1R的相互作用能为599.253 kcal.mol 1,较天然PTH(1 34)的554.083 kcal.mol 1增强7.5%;氢键数目由32对增加至38对;PTH(1 34)-(RKK-QEL)能显著刺激UAMS-32P细胞RANKL基因的表达(P<0.01),并抑制OPG基因的表达(P<0.01);PTH(1 34)-(RKK-QEL)作用于UAMS-32P和小鼠原代股骨骨髓细胞共培养体系时,能显著刺激破骨细胞的形成(P<0.01),并且活性高于PTH(1 34)标准品。     

杜仲对大鼠骨髓基质细胞骨桥蛋白和骨保护素表达的影响:水提液与醇提液有区别吗？

背景：以往实验证实杜仲能促进骨髓基质细胞向成骨细胞分化,但对促进成骨分化的分子机制报道很少。 目的：观察中药杜仲水/醇提取物对大鼠骨髓基质细胞骨桥蛋白和骨保护素表达的影响 方法：盐杜仲2 g,分别加蒸馏水或甲醇至15 mL,室温振荡1 h,静置过夜后,15 000 r/min离心10 min,弃沉淀。水浸上清不经处理即得水提取物;甲醇浸上清经真空浓缩,再加水至15 mL溶解剩余物后,得醇提取物水、醇提取物,用0.22 μm滤膜过滤后于-20 ℃保存。取2月龄SD 大鼠6 只,体外培养至第3代SD大鼠骨髓基质细胞进行杜仲水/醇提取物诱导,诱导物分为1×10-2,1×10-3,1×10-4和1×10-5 4个稀释度;阴性对照组加入PBS;诱导培养6 d。以免疫细胞化学法测定诱导6 d的骨髓基质细胞骨桥蛋白和骨保护素表达。应用反转录-聚合酶链反应方法测定1×10-3,1×10-4稀释度诱导3 d的骨髓基质细胞骨桥蛋白和骨保护素的基因表达。 结果与结论：细胞免疫化学显示,杜仲水/醇提取物对大鼠骨髓基质细胞骨桥蛋白的表达均有显著的刺激作用,以 1×10-4的稀释度作用最强,且醇提取物的作用大于水提取物;骨保护素的表达却无显著变化。反转录-聚合酶链反应显示,水提取物对诱导3 d后大鼠骨髓基质细胞骨桥蛋白的基因表达量显著高于醇提取物,骨保护素未检测出表达。证实杜仲水/醇提取物诱导骨髓基质细胞成骨分化与刺激骨桥蛋白表达相关,与骨保护素无关;水提取物促进骨髓基质细胞骨桥蛋白表达早于醇提取物。     

Full length amelogenin binds to cell surface LAMP-1 on tooth root/periodontium associated cells

Objectives

Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific amelogenin isoform, leucine-rich amelogenin peptide or LRAP. However, it is unclear if LAMP-1 is an amelogenin receptor for dental mesenchymal cells. The goal of this study was to determine if LAMP-1 serves as a cell surface binding site for full length amelogenin on tooth root/periodontium associated mesenchymal cells.

Design

Murine dental follicle cells and cementoblasts (OCCM-30) were cultured for 2 days followed by addition of full length recombinant mouse amelogenin, rp(H)M180. Dose-response (0-100 μg/ml) and time course (0-120 min) assays were performed to determine the optimal conditions for live cell surface binding using immunofluorescent microscopy. A competitive binding assay was performed to determine binding specificity by adding Emdogain^® (1 mg/ml) to the media. An antibody against LAMP-1 was used to detect the location of LAMP-1 on the cell surface and the pattern was compared to cell surface bound amelogenin. Both amelogenin and cell surface LAMP-1 were immuno-co-localized to compare the amount and distribution pattern.

Results

Maximum surface binding was achieved with 50 μg/ml of rp(H)M180 for 120 min. This binding was specific as demonstrated by competitive inhibition (79% lower) with the addition of Emdogain^®. The binding pattern for rp(H)M180 was similar to the distribution of surface LAMP-1 on dental follicle cells and cementoblasts. The high co-localization coefficient (0.92) for rp(H)M180 and LAMP-1 supports rp(H)M180 binding to cell surface LAMP-1.

Conclusions

The data from this study suggest that LAMP-1 can serve as a cell surface binding site for amelogenin on dental follicle cells and cementoblasts.     

Alterations in the immuno-skeletal interface drive bone destruction in HIV-1 transgenic rats

Osteoporosis and bone fractures are increasingly recognized complications of HIV-1 infection. Although antiretroviral therapy itself has complex effects on bone turnover, it is now evident that the majority of HIV-infected individuals already exhibit reduced bone mineral density before therapy. The mechanisms responsible are likely multifactorial and have been difficult to delineate in humans. The HIV-1 transgenic rat recapitulates many key features of human AIDS. We now demonstrate that, like their human counterparts, HIV-1 transgenic rats undergo severe osteoclastic bone resorption, a consequence of an imbalance in the ratio of receptor activator of NF-κB ligand, the key osteoclastogenic cytokine, to that of its physiological decoy receptor osteoprotegerin. This imbalance stemmed from a switch in production of osteoprotegerin to that of receptor activator of NF-κB ligand by B cells, and was further compounded by a significantly elevated number of osteoclast precursors. With the advancing age of individuals living with HIV/AIDS, low bone mineral density associated with HIV infection is likely to collide with the pathophysiology of skeletal aging, leading to increased fracture risk. Understanding the mechanisms driving bone loss in HIV-infected individuals will be critical to developing effective therapeutic strategies.     

女性血清卵泡刺激素水平与护骨素、瘦素、TGF-β1及 TGF-β2之间的关系

目的：了解女性卵泡刺激素（follicle stimulating hormone,FSH）水平与骨代谢密切相关的几种细胞因子之间的关系。方法：测量703例年龄为20~80岁健康女性的血清FSH、护骨素（osteoprotegerin,OPG）、瘦素（leptin）、转化生长因子（transforming growth factor,TGF)β1和β2,并分析它们之间的关系。结果：血清FSH与OPG(r=0.447, P＜0.01)和TGF-β2 (r=0.344, P=＜0.01)呈正相关,与TGF-β1呈负相关(r=-0.374, P＜0.01);经年龄调整后血清FSH与瘦素呈负相关(r=-0.265, P＜0.01)。多元线性回归分析显示,FSH对TGF-β1是一个负性决定因素,其决定性作用最大为22.6%;FSH对OPG和TGF-β2是一个正性决定因素,其决定性作用分别为9.9%和1.1%。FSH对瘦素几乎无影响。结论：女性年龄相关的FSH水平与血液中细胞因子TGF-β1,OPG和TGF-β2的变化有关。     

p38MAPK抑制对成骨细胞核因子-κB受体激活配体和骨保护素表达的影响

 目的 观察p38MAPK信号转导通路在成骨细胞分化及核因子-κB受体激活配体（receptor activator of nuclear factor-κB ligand,RANKL）和骨保护素（osteoprotegerin,OPG）表达中的作用。方法 取第1继代BALB/c小鼠颅盖骨成骨细胞,药物刺激组分别加入10^-8 mol/L 17β-雌二醇和10^-7 mol/L雷洛昔芬;含阻断剂组预先添加5 μmol/L SB202190阻断p38MAPK通路后,再加17β-雌二醇或雷洛昔芬。72 h后用PNPP法测定成骨细胞内碱性磷酸酶（alkaliphosphatase,ALP）活性;RT-PCR法检测成骨细胞ALP、OPG和RANKL的转录水平。结果 17β-雌二醇和雷洛昔芬能够促进成骨细胞分化,促进OPG、RANKL的表达（P<0.05）,OPG/RANKL无明显变化（P>0.05）;阻断p38MAPK信号转导通路后,成骨细胞分化受抑,OPG、RANKL的表达和OPG/RANKL降低（P<0.05）。结论 p38MAPK抑制可干扰体外培养成骨细胞分化,抑制RANKL和OPG的过度表达。