RANKL、RANK蛋白在胶质母细胞瘤组织中的表达及临床意义

目的:分析RANKL、RANK蛋白在胶质母细胞瘤组织中的表达及临床意义。方法:收集2017年10月至2019年10月于我科行手术治疗的21例胶质母细胞瘤患者的临床资料，用蛋白印迹试验检测肿瘤中心组织、瘤周组织、皮层造瘘脑组织中RANKL、RANK表达情况，并对其进行统计学分析。结果:肿瘤中心组织RANKL表达高于瘤周组织，有统计学差异(P=0.032)；瘤周组织高于皮层造瘘脑组织，有统计学差异(P=0.024)。瘤周组织RANK表达明显高于肿瘤中心组织，有统计学差异(P=0.006)；肿瘤中心组织高于皮层造瘘脑组织，有统计学差异(P=0.028)。结论:肿瘤中心组织中RANKL表达高于瘤周组织、脑组织，而瘤周组织中RANK明显高于肿瘤中心组织、肿瘤中心组织高于脑组织，我们认为RANKL/RANK通路可能在胶质母细胞瘤侵袭过程中起重要作用，有待后续进一步试验。     

从中医“虚”论治原发性骨质疏松症与OPG/RANK/RANKL信号通路的相关性

原发性骨质疏松症(primary osteoporosis,POP)是现代临床中常见的骨科疾病,其归属于中医学之"骨痿""骨痹""骨枯"等范畴。随着中医药事业的发展,中医学在POP的治疗中应用愈加广泛,且疗效显著,已受到学界的广泛认可。但遗憾的是,其缺乏现代研究理论依据的支撑。随着分子生物学的发展,OPG/RANK/RANKL信号通路的发现对中医学治疗此病具有重要意义。故笔者将从中医"虚"理论出发,并以中医学脏腑辨证理论为基础,借助现代分子生物学的研究成果OPG/RANK/RANKL信号通路,揭示中医治疗POP的OPG/RANK/RANKL信号通路表达机制,以期为中医学治疗POP提供科学理论依据。     

Low-energy laser stimulates tooth movement velocity via expression of RANK and RANKL

OBJECTIVE: Recent studies have demonstrated that low-energy laser irradiation stimulates bone formation in vitro and in vivo. However, very little is known about the effects of laser irradiation on osteoclastogenesis. The receptor activator of the nuclear factor-kB (RANK) / RANK ligand (RANKL) / osteoprotegerin (OPG) system is essential and sufficient for osteoclastogenesis. The present study was designed to examine the effects of low-energy laser irradiation on expressions of RANK, RANKL, and OPG during experimental tooth movement. DESIGN: To induce experimental tooth movement in rats, 10 g of orthodontic force was applied to the molars. Next, a Ga-Al-As diode laser was used to irradiate the area around the moved tooth and the amount of tooth movement was measured for 7 days. Immunohistochemical staining with RANK, RANKL, and OPG was performed. Real time PCR was also performed to elucidate the expression of RANK in irradiated rat osteoclast precursor cells in vitro. RESULTS: In the irradiation group, the amount of tooth movement was significantly greater than in the non-irradiation group by the end of the experimental period. Cells that showed positive immunoreactions to the primary antibodies of RANKL and RANK were significantly increased in the irradiation group on day 2 and 3, compared with the non-irradiation group. In contrast, the expression of OPG was not changed. Further, RANK expression in osteoclast precursor cells was detected at an early stage (day 2 and 3) in the irradiation group. CONCLUSION: These findings suggest that low-energy laser irradiation stimulates the velocity of tooth movement via induction of RANK and RANKL.     

Panostotic Expansile Bone Disease With Massive Jaw Tumor Formation and a Novel Mutation in the Signal Peptide of RANK

Precise regulation of bone resorption is critical for skeletal homeostasis. We report a 32‐year‐old man with a panostotic expansile bone disease and a massive hemorrhagic mandibular tumor. Originally from Mexico, he was deaf at birth and became bow‐legged during childhood. There was no family history of skeletal disease. Puberty occurred normally, but during adolescence he experienced difficulty straightening his limbs, sustained multiple fractures, and developed a bony tumor on his chin. By age 18 years, all limbs were misshapen. The mandibular mass grew and protruded from the oral cavity, extending to the level of the lower ribs. Other bony defects included a similar maxillary mass and serpentine limbs. Upon referral at age 27 years, biochemical studies showed serum alkaline phosphatase of 1760 U/L (Nl: 29‐111) and other elevated bone turnover markers. Radiography of the limbs showed medullary expansion and cortical thinning with severe bowing. Although the jaw tumors were initially deemed inoperable, mandibular mass excision and staged partial maxillectomy were eventually performed. Tumor histopathology showed curvilinear trabeculae of woven bone on a background of hypocellular fibrous tissue. Fibrous dysplasia of bone was suspected, but there was no mutation in codon 201 of GNAS in samples from blood or tumor. His clinical and radiographic findings, elevated serum markers, and disorganized bone morphology suggested amplified receptor activator of NF‐κB (RANK) signaling, even though his disorder differed from conditions with known constitutive activation of RANK signaling (eg, familial expansile osteolysis). We found a unique 12‐base pair duplication in the signal peptide of TNFRSF11A, the gene that encodes RANK. No exon or splice site mutations were found in the genes encoding RANK ligand or osteoprotegerin. Alendronate followed by pamidronate therapies substantially decreased his serum alkaline phosphatase activity. This unique patient expands the phenotypes and genetic basis of the mendelian disorders of RANK signaling activation. © 2014 American Society for Bone and Mineral Research.     

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.     

RANK-dependent autosomal recessive osteopetrosis: characterization of five new cases with novel mutations

Autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder attributed to reduced bone resorption by osteoclasts. Most human AROs are classified as osteoclast rich, but recently two subsets of osteoclast-poor ARO have been recognized as caused by defects in either TNFSF11 or TNFRSF11A genes, coding the RANKL and RANK proteins, respectively. The RANKL/RANK axis drives osteoclast differentiation and also plays a role in the immune system. In fact, we have recently reported that mutations in the TNFRSF11A gene lead to osteoclast-poor osteopetrosis associated with hypogammaglobulinemia. Here we present the characterization of five additional unpublished patients from four unrelated families in which we found five novel mutations in the TNFRSF11A gene, including two missense and two nonsense mutations and a single-nucleotide insertion. Immunological investigation in three of them showed that the previously described defect in the B cell compartment was present only in some patients and that its severity seemed to increase with age and the progression of the disease. HSCT performed in all five patients almost completely cured the disease even when carried out in late infancy. Hypercalcemia was the most important posttransplant complication. Overall, our results further underline the heterogeneity of human ARO also deriving from the interplay between bone and the immune system, and highlight the prognostic and therapeutic implications of the molecular diagnosis.     

Gingival Crevicular Fluid Levels of Sclerostin,Osteoprotegerin, and Receptor Activator of Nuclear Factor‐κB Ligand in Periodontitis

Background: To investigate changes in the levels and relative ratios of sclerostin, osteoprotegerin (OPG), and receptor activator of nuclear factor‐κB ligand (RANKL) in the gingival crevicular fluid (GCF) of patients with periodontitis after non‐surgical periodontal treatment. Methods: Fifty‐four individuals (27 healthy controls and 27 patients with chronic periodontitis [CP]) were enrolled in the study. Periodontitis patients received non‐surgical periodontal therapy. GCF sampling and clinical periodontal parameters were assessed before and 6 weeks after therapy. Sclerostin, OPG, and RANKL levels were measured by enzyme‐linked immunosorbent assay, and their relative ratios were calculated. Results: Total amounts and concentrations of sclerostin were significantly higher in patients with CP than in healthy individuals (P <0.025) and decreased after treatment (P <0.05). The RANKL/OPG ratio was significantly lower in healthy individuals than in patients with periodontitis before and after treatment (P <0.025), but no significant difference was observed in patients with periodontitis after treatment (P >0.05). The sclerostin/OPG and sclerostin/RANKL ratios were significantly lower in healthy individuals than in patients with periodontitis before and after treatment (P <0.025) and decreased in patients with periodontitis after treatment (P <0.05). Conclusions: The GCF sclerostin level may be more reliable than the RANKL/OPG ratio as a diagnostic and prognostic marker of periodontal disease and treatment outcome. Regulation of sclerostin levels may aid the development of new therapeutic strategies for the treatment of periodontal disease.     

补骨脂提取物干预骨质疏松模型大鼠骨密度及骨生物力学的变化

文题释义： RANKL/OPG通路：核因子κB受体活化因子配体(receptor activator of NF-κB Ligand,RANKL)/骨保护蛋白在维持成骨与破骨的动态平衡,调节骨代谢功能方面发挥着重要作用。RANKL 可与破骨细胞前体细胞膜表面的核因子κB受体活化因子结合,促进破骨细胞分化和激活,抑制其凋亡,最终促进骨吸收;骨保护蛋白作为RANKL的天然抗体,可与RANKL直接结合,竞争性抑制核因子κB受体活化因子与其的结合,进而抑制破骨细胞分化、成熟,最终抑制骨吸收。生理状态下,体内RANKL与骨保护蛋白的表达保持一定的比例,若二者表达比例失衡,则会造成骨代谢紊乱,导致骨相关疾病发生。 骨生物力学：是骨组织在外力作用下的力学生物学效应,对其进行检测可直接评价骨质量,是评价各种治疗骨丢失措施的最佳方案,其检测指标包括弹性模量、最大载荷、屈服载荷等。 背景：地塞米松作为一种糖皮质激素,长期应用会破坏成骨与破骨的动态平衡,降低骨密度,损伤骨生物力学,调控核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)/骨保护蛋白通路可能影响地塞米松诱导的骨质疏松症大鼠骨密度及骨生物力学。 目的：探讨基于RANKL/骨保护蛋白通路研究补骨脂提取物对地塞米松诱导骨质疏松症大鼠骨密度及骨生物力学的影响。 方法：SPF级Wistar大鼠肌肉注射地塞米松,建立骨质疏松大鼠模型。选择1×10⁷ TU/mL浓度的慢病毒载体进行实验。将模型大鼠随机分为模型组、空载组(空慢病毒载体)、骨保护蛋白沉默组(含骨保护蛋白基因干扰片段的慢病毒载体)、补骨脂提取物组、补骨脂提取物+骨保护蛋白沉默组,每组12只,另取12只正常大鼠设为对照组。药物处理后,采用骨密度仪测定大鼠左侧股骨骨密度,采用力学实验测试机测定大鼠右侧股骨生物力学指标弹性模量、最大载荷、屈服载荷,测定大鼠股骨骨矿物盐含量,酶联免疫吸附法检测血清中RANKL、骨保护蛋白水平,蛋白免疫印迹法检测骨组织中RANKL、骨保护蛋白表达水平。实验方案经青海大学医学院动物实验伦理委员会批准(批准号为2017081501) 结果与结论：①大鼠骨密度、弹性模量、最大载荷、屈服载荷、骨矿物盐含量、血清中骨保护蛋白水平、骨组织中骨保护蛋白表达：模型组较对照组降低,骨保护蛋白沉默组较模型组降低,补骨脂提取物组较模型组升高,补骨脂提取物+骨保护蛋白沉默组较骨保护蛋白沉默组升高,较补骨脂提取物组降低(均P < 0.05);②大鼠血清中RANKL水平、骨组织中RANKL蛋白表达结果显示,模型组较对照组升高;骨保护蛋白沉默组较模型组升高,补骨脂提取物组较模型组降低,补骨脂提取物+骨保护蛋白沉默组较骨保护蛋白沉默组降低,较补骨脂提取物组升高(均P < 0.05);③模型组与空载组两组间各指标比较无明显变化(P > 0.05);④结果说明,补骨脂提取物可提高地塞米松诱导骨质疏松症大鼠的骨密度,改善其骨生物力学,可能是通过上调骨保护蛋白表达,下调RANKL表达实现的。 ORCID: 0000-0001-9896-6214(周倚墨) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Hypoxia mimetic deferoxamine influences the expression of histone acetylation- and DNA methylation-associated genes in osteoblasts

Purpose of the study: Sufficient oxygen supply to bone tissue is essential for normal bone development and efficient bone repair. Hypoxia and hypoxia-inducible factor 1α (HIF1α) signaling pathway have been shown to exhibit profound effects on proliferation, differentiation as well as gene and protein expression in osteoblasts, osteoclasts and mesenchymal stem cells; however, as epigenetic mechanisms also perform an important regulatory role in these cells, our aim was to elucidate whether hypoxia mimetic deferoxamine could influence epigenetic mechanisms in bone cells by modulating the gene expression levels of chromatin-modifying enzymes.

Materials and methods: Osteoblast cell line HOS was exposed to deferoxamine, a widely used hypoxia mimetic, and expression profile of 40 genes associated with histone acetylation, deacetylation and DNA methylation was determined using quantitative real time polymerase chain reaction (qPCR) array followed by individual qPCR analyses. In addition, genes associated with hypoxia response, RANK/RANKL/OPG system, WNT/β-catenin signaling pathway and oxidative stress were also analyzed.

Results: We observed induced expression of histone deacetylase 9 (HDAC9) and suppressed expression of K(lysine) acetyltransferase 5 (KAT5) and DNA methyltransferase 3A (DNMT3A) demonstrating for the first time that expression of genes encoding chromatin-modifying enzymes could be influenced by hypoxia mimetic in HOS cells.

Conclusions: Based on our results we can conclude that hypoxia mimetic deferoxamine influences expression of histone acetylation- and DNA methylation-associated genes in osteoblasts and that further studies of hypoxia-induced epigenetic changes in bone cells should be undertaken.