Wnt信号通路与骨细胞生物力刺激信号转导间的关系

Wnt信号通路是体内重要的信号调节系统之一,而骨细胞作为骨组织中最主要的应力感受细胞,受载后可引起其内部Wnt信号通路的快速激活,最终调节其基因表达,影响骨改建。该激活过程受诸多调控因子的影响。本文就Wnt信号通路与骨细胞生物力刺激信号转导间的关系,以及该信号转导过程中Wnt信号通路的调控因子等研究进展作一综述。     

BmaI1在经典Wnt通路调控间充质干细胞衰老中的作用

目的： 探讨Bmal1与Wnt经典信号通路对间充质干细胞（mesenchymal stem cells,MSCs）的衰老是否具有协同或拮抗作用。方法： 分为转染组和空白对照组,转染组用Bmal1的慢病毒转染NIH-3T3细胞表达增强后,通过实时荧光定量PCR和Western免疫印迹等方法检测转染组和空白组β-catenin、TCF1表达量。采用SPSS 19.0软件包对数据进行统计学分析。结果： 经PCR鉴定,Bmal1基因重组慢病毒载体构建成功,转染Bmal1表达增强后,β-catenin表达也显著增强（P<0.05）,但与TCF1增强比例不同;而TCF1的变化无统计学意义。结论： Bmal1对Wnt 经典信号通路直接或间接调控小鼠 BMSCs 的增龄性改变具有协同作用。     

经典Wnt信号通路与牙周膜干细胞成骨分化

牙周炎是口腔最常见的疾病之一,累及牙周支持组织,随着疾病的进展将引起附着丧失、牙周袋形成、牙槽骨吸收,最终导致牙齿松动脱落。因被牙周炎破坏吸收的牙槽骨自愈能力十分有限,所以牙周炎的治疗目标是在彻底清除菌斑生物膜的基础上,争取获得较多的牙周组织再生。牙周膜干细胞作为最适宜进行牙周组织再生的细胞,被广泛研究。Wnt信号通路分为经典Wnt通路和非经典Wnt信号通路,为十分复杂而高度保守的通路传导途径。该通路与牙周膜干细胞成骨分化的关系十分密切,牙周膜干细胞的成骨分化又对牙周组织再生有重要意义。该文对经典Wnt信号通路与牙周膜干细胞成骨分化的研究概况作一综述。     

Wnt信号通路相关基因在颌骨牙源性角化囊性瘤中的表达及意义

目的研究Wnt信号通路相关基因在颌骨牙源性角化囊性瘤中的表达,探讨其发挥的调控作用。方法收集颌骨牙源性角化囊性瘤新鲜标本及同一患者的正常口腔黏膜,提取RNA,基因芯片检测Wnt信号通路的相关基因,进行生物信息学分析。结果颌骨牙源性角化囊性瘤与正常口腔黏膜相比,在Wnt信号通路相关基因中发现5个共同差异表达基因,其中CAMK2A下调, FZD3、MAPK10、PRKX、WNT5a上调。结论牙源性角化囊性瘤中存在Wnt信号通路基因的异常表达,Wnt信号通路相关基因在颌骨牙源性角化囊性瘤中发挥一定的调控作用。     

MicroRNA-27b inhibits cell proliferation in oral squamous cell carcinoma by targeting FZD7 and Wnt signaling pathway

This study intended to investigate the role of microRNA-27b (miR-27b) in proliferation of oral squamous cell carcinoma (OSCC) cells and to explore the potential molecular mechanism. Cell proliferation was detected by MTT assay. The expression levels of miR-27b, Frizzled7 (FZD7), cyclin D1 and c-myc were detected by quantitative real time polymerase chain reaction (qRT-PCR). The protein expression level of FZD7 was detected by western blot analysis. The relationship between miR-27b and FZD7, and the activity of Wnt signaling pathway were determined using luciferase reporter assay. The miR-27b expression in OSCC cell lines was significantly decreased compared with control. Overexpression of miR-27b remarkably inhibited OSCC cell proliferation. Additionally, miR-27b could target and inhibit FZD7 expression and decrease the activity of Wnt signaling pathway.miR-27b could inhibit OSCC cell proliferation through inhibiting FZD7 and FZD7-mediated Wnt signaling pathway.     

Mutational analysis of Wnt signaling molecules in ameloblastoma with aberrant nuclear expression of β‐catenin

Background: To clarify the genetic background of ameloblastoma, expression of β‐catenin, and mutational status of genes involved in Wnt signaling pathway were investigated. Methods: We analyzed β‐catenin and cyclin D1 in 18 cases of ameloblastoma by immunohistochemical staining, and searched for mutations in CTNNB1 (gene for β‐catenin), APC, AXIN1, and AXIN2 by polymerase chain reaction (PCR) and direct sequencing method. Result: We detected membranous and occasionally cytoplasmic expression of β‐catenin in 16 of 18 cases (89%), and nuclear expression of β‐catenin principally in the peripheral columnar cells in 11 of 18 cases (61%). In nine of the 18 cases (50%), we detected the expression of cyclin D1 principally in the peripheral columnar cells. However, there was no correlation between nuclear expressions of β‐catenin and cyclin D1. No missense mutations were found in CTNNB1, APC, AXIN1, and AXIN2 in all cases except for silent mutation and already‐known single nucleotide polymorphism. Conclusion: Mutations in CTNNB1, APC, AXIN1, and AXIN2 are not implicated in nuclear accumulation of β‐catenin, and that the expression of cyclin D1 is accelerated independently of β‐catenin in ameloblastomas. Other Wnt signaling members or alternative pathways involved in the degradation of β‐catenin should be subject of further investigation.     

Differential expression of canonical and non-canonical Wnt ligands in ameloblastoma

J Oral Pathol Med (2012) 41 : 332–339 Background: Canonical and non‐canonical Wnt signaling pathways modulate diverse cellular processes during embryogenesis and post‐natally. Their deregulations have been implicated in cancer development and progression. Wnt signaling is essential for odontogenesis. The ameloblastoma is an odontogenic epithelial neoplasm of enamel organ origin. Altered expressions of Wnts‐1, ‐2, ‐5a, and ‐10a are detected in this tumor. The activity of other Wnt members remains unclarified. Materials and Methods: Canonical (Wnts‐1, ‐2, ‐3, ‐8a, ‐8b, ‐10a, and ‐10b), non‐canonical (Wnts‐4, ‐5a, ‐5b, ‐6, 7a, ‐7b, and ‐11), and indeterminate groups (Wnts‐2b and ‐9b) were examined immunohistochemically in 72 cases of ameloblastoma (19 unicystic [UA], 35 solid/multicystic [SMA], eight desmoplastic [DA], and 10 recurrent [RA]). Results: Canonical Wnt proteins (except Wnt‐10b) were heterogeneously expressed in ameloblastoma. Their distribution patterns were distinctive with some overlap. Protein localization was mainly membranous and/or cytoplasmic. Overexpression of Wnt‐1 in most subsets (UA = 19/19; SMA = 35/35; DA = 5/8; RA = 7/10) (P < 0.05), Wnt‐3 in granular cell variant (n = 3/3), and Wnt‐8b in DA (n = 8/8) was key observations. Wnts‐8a and ‐10a demonstrated enhanced expression in tumoral buddings and acanthomatous areas. Non‐canonical and indeterminate Wnts were absent except for limited Wnt‐7b immunoreactivity in UA (n = 1/19) and SMA (n = 1/35). Stromal components expressed variable Wnt positivity. Conclusion: Differential expression of Wnt ligands in different ameloblastoma subtypes suggests that the canonical and non‐canonical Wnt pathways are selectively activated or repressed depending on the tumor cell differentiation status. Canonical Wnt pathway is most likely the main transduction pathway while Wnt‐1 might be the key signaling molecule involved in ameloblastoma tumorigenesis.     

EphrinB2 signaling enhances osteogenic/odontogenic differentiation of human dental pulp stem cells

ObjectiveTo investigate the role of the EphrinB2 signaling pathway in the osteogenesis/odontogenesis of human dental pulp stem cells (DPSCs).DesignThe endogenous expression levels of EphrinB2 and its cognate receptors EphB2 and EphB4 in DPSCs were analyzed by qRT-PCR and Western blotting after 7, 14 and 21 days of osteogenic/odontogenic induction culture. Additionally, the phosphorylation of EphrinB2, EphB4 and ERK1/2 proteins at early time-points following osteogenic induction, were also investigated by Western blots. Subsequently, we investigated whether supplementation of recombinant EphrinB2-Fc within the induction milieu can enhance the osteogenic/odontogenic differentiation of DPSCs.ResultsEndogenous gene and protein expression levels of EphrinB2, EphB2 and EphB4 were upregulated in induced versus non-induced DPSCs, over 21 days of osteogenic/odontogenic induction. Western blots showed increase in phosphorylated EphrinB2, EphB4 and ERK1/2 proteins at early time-points following osteogenic induction. Preliminary investigation of a concentration range (0, 0.5, 1 and 2 μg/ml) of recombinant EphrinB2-Fc within osteogenic induction media, showed that 0.5 μg/ml was optimal for enhancing the osteogenic/odontogenic differentiation of DPSCs over a culture duration of 14 days. Subsequently, more comprehensive qRT-PCR analysis with 0.5 μg/ml EphrinB2-Fc revealed significant upregulation of several key osteogenic marker genes in treated versus untreated DPSCs after 21 days of osteogenic/odontogenic induction. By 7 days of osteogenic induction, DPSCs treated with 0.5 μg/ml EphrinB2-Fc exhibited significantly more calcium mineralization (Alizarin red S staining) and alkaline phosphatase activity than the untreated control.ConclusionsEphrinB2 signaling plays a key role in the osteogenic/odontogenic differentiation of DPSCs.     

糖基化终末产物对牙周膜干细胞增殖及其Wnt经典信号通路相关基因表达的影响

目的:观察糖基化终末产物(AGEs)对人牙周膜干细胞(HPDLSC)增殖及其Wnt经典信号通路相关基因DKK-1、β-catenin表达的影响。方法:体外组织块法和有限稀释法克隆化培养HPDLSC,并与100μg/mL AGEs共同培养,通过细胞克隆形成率、细胞生长曲线观察AGEs对HPDLSC增殖能力的影响;RT-PCR和Western Blot检测实验组和对照组DKK-1、β-catenin mRNA以及核蛋白β-catenin的表达量。结果:经100μg/mL AGEs刺激的HPDLSC,其克隆形成率、增殖速度以及β-catenin mRNA和核蛋白β-catenin的表达量均明显低于对照组(P<0.05);而DKK-1 mRNA则明显高于对照组(P<0.05)。结论:AGEs能抑制HPDLSC的增殖及其Wnt经典信号通路。     

Hydroxyapatite-based cements induce different apatite formation in radicular dentin

ObjectiveTo investigate crystallinity and ultrastructure of the formed hydroxyapatite at radicular cervical and apical dentin after being treated with three different canal sealers.MethodsCervical and apical root dentin surfaces were treated with two experimental hydroxyapatite-based sealers, containing sodium hydroxide (calcypatite) or zinc oxide (oxipatite) and an epoxy resin-based canal sealer (AH Plus); gutta-percha without sealer was included as control. Dentin surfaces were studied by X-ray diffraction and transmission electron microscopy through selected area diffraction and bright-field imaging after 24 h and 12 m of storage.ResultsRoot cervical dentin treated with calcypatite and oxipatite produced poor crystallinity of new minerals, wide amorphous phase and non-stoichiometry. Reflections at the 002 plane and the corresponding diffraction rings attained lower values in the Scherrer equation and the Scherrer-Wilson equation in samples treated with both HAp-based sealers than in specimens without sealer or with AH Plus. At root cervical dentin treated with calcypatite, shorter and wider crystallite size formations and lower crystals grain size were found, if compared to those encountered at oxipatite treated dentin. Oxipatite attained improved crystallographic atomic order and less structural variation in both distances and angles. Apical dentin treated with oxipatite attained preferred grain orientation with polycrystalline lattices.SignificanceThe immature crystallites formed in dentin treated with calcypatite and oxipatite will account for high hydroxyapatite solubility and remineralizing activity. New polycrystalline formations encountered in apical dentin treated with oxipatite may also produce high mechanical performance.     

Roles of non-canonical Wnt signaling pathways in bone resorption

Background

Wnt is a cytokine involved in the development and homeostasis of various organs. In 2001, low-density lipoprotein receptor-related protein 5 (LRP5) was identified as the gene responsible for osteoporosis pseudoglioma syndrome and regulation of bone mass. Since LRP5 belongs to the low-density lipoprotein receptor family, this finding garnered the attention of researchers in the bone, mineral, and Wnt research fields. The role of Wnt in bone formation and resorption has since been extensively studied. Wnt/β-catenin signals are known to play a role in bone resorption. The activation of these signals in osteoblast lineage cells such as osteoblasts and osteocytes induces the expression of osteoprotegerin and then inhibits osteoclast formation.

O-GlcNAc通过抑制Wnt信号通路影响上唇发育的实验研究

目的：探讨氧联乙酰葡萄糖胺( O-linked N-acetylglucosamine,O-GlcNAc)影响上唇发育时上皮层的消失过程中,Wnt信号通路在其中发挥的作用。方法通过在鸡胚唇部植入浸有O-GlcNAc促进剂氧联乙酰葡萄糖胺转移酶(O-GlcNAc transferase,OGT)、O-GlcNAc 的抑制酶氧联乙酰葡萄糖胺水解酶(O-GlcNAcase,OGA)以及OGA的抑制剂链脲佐菌素( streptozoticin,SZT)的琼脂糖微球,构建O-GlcNAc高表达与低表达以及反向高表达实验组,并继续孵育24 h,茜素红—阿利新蓝骨组织染色观察胚胎骨、软骨发生的变化,TUNEL法检测细胞凋亡情况,免疫组化染色法检测Wnt信号系包括GSK-3β、β-catenin在内的一系列信号因子。结果 O-GlcNAc过表达组OGT和STZ中,上皮层的消失出现延迟,骨染色发现胚胎唇部出现骨缺损,TUNEL检测与正常组相比未发现明显细胞凋亡,但GSK-3β在细胞质内表达增加,β-catenin细胞核内表达减少;正常侧及O-GlcNAc 低表达组OGA中上唇发育未受影响。结论 O-GlcNAc过表达时引起上皮层的消失发生延迟,这并非由细胞凋亡减少引起,而是可能通过抑制Wnt信号通路,导致 GSK-3β、β-catenin在内的相关信号因子变化,这些变化将影响上唇发育并可能导致唇腭裂的发生。     

人牙本质磷蛋白对小型猪修复性牙本质形成作用的实验研究

目的　观察人牙本质磷蛋白 (dentinphosphoprotein ,DPP)对修复性牙本质形成的诱导作用 ,进一步研究DPP的功能。方法　用人DPP粉作盖髓剂 ,对小型猪健康恒牙进行盖髓实验 ,对照组采用氢氧化钙和空白对照 (氧化锌丁香油糊剂盖髓 ) ,通过组织病理学观察牙本质桥的形成。结果DPP盖髓术后 2周 ,穿髓孔下固有牙髓细胞分化为类造牙本质细胞 ,分布在已形成的修复性牙本质团块周围 ;术后 1个月有完整的修复性牙本质桥形成 ;术后 3个月修复性牙本质桥增厚且结构致密 ,主要是管样牙本质 ,其下方排列分化好的造牙本质细胞。氢氧化钙盖髓组 ,术后 2周界面有炎症坏死区 ,只有少量的钙质团块形成 ,1～ 3个月逐渐形成完整的修复性牙本质桥 ,但较DPP盖髓后形成修复性牙本质的速度慢且量较少。结论　人DPP对牙髓刺激性小 ,可以直接诱导牙髓细胞分化及修复性牙本质形成 ,提示DPP在牙本质发育和修复的生物矿化中起重要作用。     

靶向Wnt信号通路在牙周炎治疗领域的研究现状

牙周炎是由菌斑细菌引起并最终导致牙周支持组织吸收的感染性疾病,其药物治疗主要分为抑制炎症反应和介导牙周组织修复两个方面。 Wnt信号通路是调控胚胎干细胞以及多种组织干细胞自我更新和分化的关键途径,被认为是生物体中最为重要的信号通路之一。一些研究显示,Wnt信号通路在牙周炎的发生和治疗中起到关键作用。本综述从牙周病炎症免疫反应和介导牙周膜细胞成骨分化两个方面介绍了近年来关于Wnt信号通路调控的研究成果,并比较了可能通过激活Wnt通路介导牙周膜细胞成骨分化的潜在药物,为靶向Wnt通路治疗牙周炎提供了依据。     

赖氨酸乙酰基转移酶2A通过经典Wnt通路影响牙周膜干细胞成骨分化

目的 探讨赖氨酸乙酰基转移酶2A（KAT2A）调控牙周膜干细胞（PDLSCs）成骨分化的机制。方法 分离培养来自健康志愿者的PDLSCs（H-PDLSCs）和牙周炎患者的PDLSCs（P-PDLSCs）,比较传代细胞中KAT2A基因的表达水平。采用KAT2A基因干扰H-PDLSCs,检测KAT2A基因干扰对H-PDLSCs成骨分化的影响;同时检测KAT2A干扰后对经典Wnt通路及其配体的影响,确定KAT2A基因与经典Wnt通路的上下游关系。结果 与H-PDLSCs相比,P-PDLSCs中KAT2A基因的表达下降,差异有统计学意义（P<0.05）。KAT2A基因被干扰后,PDLSCs成骨能力下降（P<0.05）,同时经典Wnt通路被激活,拮抗剂Dickkopf-1（DKK-1）表达下调;加入DKK-1后,被干扰的PDLSCs成骨分化功能恢复,而KAT2A的表达不受影响,仍维持较低水平。结论 牙周炎可导致PDLSCs中KAT2A基因表达下降,激活经典Wnt通路,抑制细胞的成骨分化。     

Effect of propolis gel on the in vitro reduction of dentin permeability

Objective

The aim of this study was to evaluate the capacity of potassium oxalate, fluoride gel and two kinds of propolis gel to reduce the hydraulic conductance of dentin, in vitro.

Material and Methods

The methodology used for the measurement of hydraulic conductance of dentin in the present study was based on a model proposed in literature. Thirty-six 1-mm-thick dentin discs, obtained from extracted human third molars were divided into 4 groups (n=9). The groups corresponded to the following experimental materials: GI-10% propolis gel, pH 4.1; GII-30% propolis gel; GIII-3% potassium oxalate gel, pH 4,1; and GIV-1.23% fluoride gel, pH 4.1, applied to the dentin under the following surface conditions: after 37% phosphoric acid and before 6% citric acid application. The occluding capacity of the dentin tubules was evaluated using scanning electron microscopy (SEM) at ×500, ×1,000 and ×2,000 magnifications. Data were analyzed statistically by two-way ANOVA and Tukey''s test at 5% significance level.

Results

Groups I, II, III, IV did not differ significantly from the others in any conditions by reducing in hydraulic conductance. The active agents reduced dentin permeability; however they produced the smallest reduction in hydraulic conductance when compared to the presence of smear layer (P<0.05). The effectiveness in reducing dentin permeability did not differ significantly from 10% or 30% propolis gels. SEM micrographs revealed that dentin tubules were partially occluded after treatment with propolis.

Conclusions

Under the conditions of this study, the application of 10% and 30% propolis gels did not seem to reduce the hydraulic conductance of dentin in vitro, but it showed capacity of partially obliterating the dentin tubules. Propolis is used in the treatment of different oral problems without causing significant great collateral effects, and can be a good option in the treatment of patients with dentin sensitivity.