骨免疫在口腔疾病中的作用研究进展

长期以来的研究表明先天免疫和后天免疫在宿主抵御外来微生物入侵的过程中起着至关重要的作用，而在这过程中也会同时造成宿主自身的组织或器官的损害。骨骼系统就是其中之一，在一些诸如类风湿性关节炎、牙周炎等疾病中，免疫系统的反应不仅起到去除外来致病菌的作用，还会同时造成骨骼系统的破坏。长期以来大部分的研究都致力于了解免疫系统对骨组织破坏的机制，而近些年来有研究开始探究骨反作用于免疫系统，由此衍生出了一个全新的研究领域——骨免疫。文章从骨免疫的机制、免疫与骨吸收作用通路以及在口腔相关疾病的作用机制等方面总结了近些年来相关的研究进展。     

基于Cyclin D1基因敲减探讨健骨颗粒促进成骨细胞增殖的机理

目的验证Cyclin D1及其下游信号蛋白对成骨细胞增殖的调控作用,探讨健骨颗粒促进成骨细胞增殖的作用机制。方法培养成骨样细胞株UMR-106,运用CRISPR/Cas9技术敲减Cyclin D1基因后,将细胞分为基因敲减+生理盐水组(组1)、基因敲减+健骨颗粒组(组2)、病毒空载体细胞(组3,阴性对照)。未行基因敲减的细胞分为正常细胞+生理盐水组(组4)和正常细胞+健骨颗粒组(组5)。分别用生理盐水血清或健骨颗粒含药血清干预各组细胞24、48、72 h,通过CCK8法检测细胞增殖情况,利用Real time PCR检测Cyclin D1、Cyclin E、CDK 2、CDK 4、Rb及E2F-1基因的表达水平。结果经Western-bolt检测表明基因敲减的成骨细胞Cyclin D1蛋白表达明显下降。CCK8法检测结果显示,与组4相比,组5增殖速度变快(P0. 05),组3增殖速度无差异(P0. 05),组1和组2增殖速度均减慢(P0. 05);与组5相比,组2增殖速度减慢(P0. 01);与组1相比,组2增殖速度无统计学意义(P0. 05)。Real time PCR检测结果显示,与组4相比,组5 Cyclin D1、CDK2、CDK4、Cyclin E、Rb及E2F-1 mRNA的表达明显增高(P0. 05或P0. 01),组3表达无统计学意义(P0. 05),组1和组2表达明显降低(P0. 05或P0. 01);与组5相比,组2增殖速度减慢(P0. 01);与组1相比,组2无统计学意义(P0. 05)。结论 Cyclin D1及其下游信号蛋白是调控成骨细胞增殖的关键因素;健骨颗粒可以通过调节Cyclin D1及其下游信号蛋白促进成骨细胞增殖。     

High-magnitude mechanical strain inhibits the differentiation of bone-forming rat calvarial progenitor cells

Purpose: Orthodontic tooth movement occurs during the bone remodeling induced by therapeutic mechanical strain. It is important to investigate the relation between the strength of mechanical stress and bone formation activity. The aim of this study was to determine the effect of high-magnitude mechanical strain on bone formation in detail.

Materials and methods: Osteoblast-like cells isolated from fetal rat calvariae were loaded with 18% cyclic tension force (TF) for 48?h. To phenotypically investigate the effect of TF, we measured the number and the size of bone nodules stained by von Kossa technique on day 21 after cell seeding and determined the calcium content of bone nodules on day 14. Furthermore, we examined the gene expression of BMP-2, Runx2 and Msx2, which are important factors for bone nodule formation, on days 1, 4 and 7 after TF loading.

Results: The maximum bone nodule size in the control group was 1620 and 719?μm in the TF group. Furthermore, the mean number of bone nodules sized over 360?μm in the TF group was significantly decreased compared to the control group. The calcium content was also significantly decreased to 42% by TF loading. The mRNA expression of BMP-2, Runx2 and Msx2 was decreased 1 and 4 days after TF loading.

Conclusion: The differentiation of bone forming progenitor cells into bone nodule forming cells was inhibited by TF due to the decreased expression of bone formation related factors such as BMP-2, Runx2 and Msx2.     

梓醇防治骨质疏松症的研究进展

骨质疏松症常易导致骨折，致残和致死率高，严重影响患者生活质量。中药地黄及组方在临床上抗骨质疏松效果显著，已有越来越多的研究发现和报道了其活性成分梓醇在这方面的作用和机制，取得了一定的研究成果。本文将通过Pubmed、Web of science、中国知网和万方数据库等对于近10年的相关文献进行检索，搜集并整理有关地黄、梓醇抗骨质疏松方面的最新研究进展，归纳梓醇通过颅骨缺损模型、卵巢切除模型和糖尿病骨质疏松模型等体内动物模型和骨髓间充质干细胞、成骨细胞和破骨细胞等体外细胞模型发挥抗骨质疏松作用的实验结果，分析其研究动态、给药剂量、作用指标以及可能影响Wnt、Hedgehog和PTEN/RANKL等相关信号通路，挖掘潜在的作用靶点和分子机制，为更好地发挥、研究地黄和梓醇抗骨质疏松的作用及机制提供借鉴。     

Functionally deficient mesenchymal stem cells reside in the bone marrow niche with M2‐macrophages and amyloid‐β protein adjacent to loose total joint implants

We sought to demonstrate whether there is a difference in the local mesenchymal stem cells (MSC) niche obtained from patients undergoing their first total joint replacement surgery versus those patients undergoing a revision surgery for an failing total joint implant. Bone marrow aspirates collected from patients undergoing revision total joint arthroplasty were observed to be less clonal and the expression of PDGFRα, CD51, ALCAM, endoglin, CXCL12, nestin, and nucleostemin were decreased. Revision MSC were also less able to commit to an osteoblast‐lineage or an adipocyte‐lineage. Further, in revision MSC, OPG, and IL6 expression were increased. Monocytes, derived from revision whole marrow aspirates, were less capable of differentiating into osteoclasts, the cells implicated in the pathologic degradation of bone. Osteoclasts were also not observed in tissue samples collected adjacent to the implants of revision patients; however, the alternatatively activated M2‐macrophage phenotype was observed in parallel with pathologic accumulations of amyloid‐β, τ‐protien and 3‐nitrotyrosine. Despite the limited numbers of patients examined, our data suggest that nucleostemin may be a useful functional marker for MSC while the observation of M2‐macrophage infiltration around the implant lays the foundation for future investigation into a novel mechanism that we propose is associated with loose total joint implants. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:615–624, 2014.     

Expression of angiopoietin‐like protein 4 at the fracture site: Regulation by hypoxia and osteoblastic differentiation

Vascular disruption that occurs as a consequence of bone fracture, leads to hypoxia at the site of damage. Hypoxia regulates the expression of a number of genes that can modulate energy conservation, cell survival, tissue regeneration and angiogenesis. In this study we investigated the expression of Angiopoietin‐like 4, an adipocytokine that has additional roles in angiogenesis, at the fracture site. We demonstrate that Angptl4 mRNA expression increased early during fracture healing (day 3) returning close to baseline at day14. In the callus, Angptl4 mRNA was visualized in areas of condensing mesenchymal cells, callus cartilage and was especially high in mineralizing osteoblasts located in areas of new bone formation. In vitro, Angptl4 mRNA expression in osteoblasts increased under hypoxic conditions and in cells treated with the hypoxia mimetic desferrioxamine. Angptl4 levels were strongly induced at day 14 in differentiating MC3T3‐E1 osteoblastic cells. Exogenous ANGPTL4 increased expression of Runx2, Spp1, vegfa, and Alp mRNA in differentiating osteoblasts. We suggest that the distribution of Angptl4 in the callus may be driven by hypoxia and that Angptl4 may play a role in osteoblastic differentiation, and possibly angiogenesis via regulation of VEGF. Further studies could reveal a dual role for Angptl4 in angiogenesis and osteogenesis. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1364–1373, 2015.     

大鼠下颌骨来源的成骨细胞对甲硝唑和替硝唑的转运

目的：应用SD大鼠下颔骨来源的原代成骨细胞，观察成骨细胞对甲硝唑和替硝唑的转运，探讨人工种植牙给药的影响及可行性。方法：将原代培养的新生鼠成骨细胞（newborn rat’s osteoblast，N）和成年鼠成骨细胞（adult rat’s osteoblast，A）与溶于PBS的药物共同孵育，分别于1，3，5，10min后，弃去细胞外液，收集各时间点细胞悬液，用高效液相色谱法测定细胞内药物量，用考马斯亮蓝法测定细胞蛋白总量。结果：（1）高效液相色谱法可以准确测定2种药物的量。（2）2种成骨细胞均可将甲硝唑和替硝唑转运至细胞内。结论：成骨细胞具有转运硝基眯唑类药物的能力，证实了人工种植牙给药的可行性。     

维生素D3衍生物对人成骨细胞生长和碱性磷酸酶活性的影响

目的：使用人胎成骨细胞（ＯＢ）为体外模型，观察了１，２５－双羟维生素Ｄ３〔１，２５（ＯＨ）２Ｄ３〕、２４，２５－双羟维生素Ｄ３［２４，２５（ＯＨ）２Ｄ３）］对ＯＢ生长和代谢的影响。方法：用ＡＬＰ测定法和Ｂｒａｄｆｏｒｄ蛋白含量法。结果：１，２５（ＯＨ）２Ｄ３在浓度为１０８ｍｏｌ／Ｌ时，刺激碱性磷酸酶（ＡＬＰ）的活性。但抑制ＯＢ的生长。２４，２５（ＯＨ）２Ｄ３在１０－８ｍｏｌ／Ｌ时无上述作用。结论：１，２５（ＯＨ）２Ｄ３对ＯＢ的ＡＬＰ有直接的促活作用，其作用与时间相关。２４，２５（ＯＨ）２Ｄ３对ＯＢ的ＡＬＰ活性无关。１，２５（ＯＨ）２Ｄ３对人胎ＯＢ生长有抑制作用。     

The role of sirtuin 1 in osteoblastic differentiation in human periodontal ligament cells

Lee Y‐M, Shin S‐I, Shin K‐S, Lee Y‐R, Park B‐H, Kim E‐C. The role of sirtuin 1 in osteoblastic differentiation in human periodontal ligament cells. J Periodont Res 2011; 46: 712–721. © 2011 John Wiley & Sons A/S Background and Objective: Activation of sirtuin 1 (SIRT1) promotes the differentiation of keratinocytes and mesenchymal stem cells, but inhibits the differentiation of muscle and fat cells. However, the involvement of SIRT1 in the differentiation of human periodontal ligament cells into osteoblast‐like cells remains unclear. To identify the role of SIRT1 in human periodontal ligament cells, we measured SIRT1 mRNA and SIRT1 protein levels during the osteoblastic differentiation of human periodontal ligament cells. Additionally, we investigated the effects of overexpressing and underexpressing SIRT1 on the differentiation of human periodontal ligament cells, and the signaling mechanisms involved. Material and Methods: Expression of SIRT1 and osteoblastic differentiation markers was assessed by RT‐PCR, real‐time PCR, Alizarin red staining and western blotting. Results: Marked upregulation of SIRT1 mRNA and SIRT1 protein was observed in cells grown for 3 d in osteogenic induction medium (OM). Activation of SIRT1 using resveratrol and isonicotinamide stimulated osteoblastic differentiation in a dose‐dependent manner, as assessed by the expression of mRNAs encoding alkaline phosphatase, osteopontin, osteocalcin, osterix and Runx2, and induced calcium deposition. In contrast, inhibition of SIRT1 using sirtinol, nicotinamide and gene silencing by RNA interference suppressed mineralization and the expression of osteoblast marker mRNAs. Further mechanistic studies revealed that resveratrol treatment increased the phosphorylation of Akt, adenosine monophosphate kinase (AMPK), Smad 1/5/8 and c‐Jun N‐terminal kinase, but reduced OM‐induced activation of nuclear factor‐κB. Conversely, application of sirtinol suppressed the phosphorylation of Akt, AMPK, Smad 1/5/8, p38, ERK and c‐Jun N‐terminal kinase, and enhanced nuclear factor‐κB activity, in OM‐stimulated cells. Conclusion: These data suggest that SIRT1 is a potent regulator of differentiation of human periodontal ligament cells and may have clinical implications for periodontal bone regeneration.