NMDA受体拮抗剂减少大鼠牙髓伤害性刺激诱导的延髓Fos …

目的：探讨谷氨酸及其ＮＭＤＡ受体是否参与牙髓伤害性刺激的传递揭示它们与牙痛传递的内在关系。方法：随机分配为４组的２８只大鼠。一组单纯给予穿髓刺激,另一组在穿髓刺激前预先侧脑室分别给予３种浓度的谷氨酸ＮＭＤＡ受体拮抗剂ＭＫ－８０１。另设ＭＫ－８０１对照组和正常对照组。     

大鼠牙髓伤害性刺激诱导延髓内Fos表达

目的：研究牙髓伤害性信息在延髓传入的相关部位。方法：用抗Ｆｏｓ蛋白的免疫组织化学方法,对大鼠牙齿机械穿髓后诱导的延髓内Ｆｏｓ表达部位进行了观察。同时结合酪氨酸羟化酶（ＴＨ）免疫组化方法,观察ＦＯＳ阳性神经元与ＴＨ阳性神经元的关系。结果：Ｆｏｓ样免疫反应神经元主要分布在同侧三叉神经脊束核尾侧亚核（Ｖｃ）、双侧延髓内脏带（孤束核、腹外侧区及二者之间的网状结构）。在抗Ｆｏｓ和抗酪氨酸羟化酶的双重免疫染色切片上,见到延髓内脏带内许多Ｆｏｓ阳性神经元的胞浆同时呈ＴＨ样免疫反应（Ｆｏｓ－ＴＨ双重阳性细胞）。结论：三叉神经脊束核尾侧亚核在牙髓伤害性信息传递过程中具有重要作用;延髓内脏带可能参与了调控牙髓暴露所致的疼痛应激反应。     

伤害性刺激大鼠切牙引起三叉神经脊束核尾侧亚核内Fos的表达

在甲氧氟烷麻醉下，给大鼠切牙不同形式的伤害性刺激．动物存活２ｈ后，用ＡＢＣ法检查大鼠三叉神经脊束核尾侧亚核神经元的Ｆｏｓ（原癌基因ｃ－ｆｏｓ表达的蛋白产物）．结果发现，几种伤害性刺激均引起同侧三叉神经脊束核尾侧亚核神经元的Ｆｏｓ表达．以机械穿髓为最多，依次是化学刺激、热刺激和电刺激．提示不同的刺激引起牙髓痛反应程度可能不同，其结果可为牙痛研究提供较为客观的指标，并可用以探索牙痛的的中枢调制和传导途径．     

Leptin受体在人牙髓干细胞中表达的实验研究

目的:研究体外培养人牙髓干细胞表型特征及生物学性状,检测Leptin受体在牙髓干细胞表面的表达.方法:采用组织块法和酶消化法分离培养人牙髓十细胞;免疫组化法、免疫荧光法检测细胞表面分子的表达;体外诱导分化实验检测细胞多向分化能力.免疫组化法检测Leptin受体在人牙髓干细胞表面的表达.结果:分离获得的牙髓干细胞波形丝蛋白和CD146表达阳性,具有向脂肪细胞和成骨细胞分化的潜能;Leptin受体在牙髓干细胞表面阳性表达.结论:分离培养的牙髓干细胞具有干细胞的表型特征及生物学性状,Leptin受体可表达于牙髓干细胞.     

人前磨牙牙髓牙本质复合体层粘连蛋白受体的免疫组织化学研究

目的：研究层粘连蛋白受体在牙髓牙本质复合体内的表达，探讨其分布与功能的关系。方法：收集新鲜健康人前磨牙60个，固定，脱钙，石蜡包埋，层粘连蛋白受体SABC免疫组织化学染色。结果：层状连蛋白受体免疫反应物（LNR－IR）普遍存在于牙髓牙本质复合体的神经纤维膜、血管内皮细胞、成牙本质 细胞及成纤维细胞上，以牙髓的神经纤维膜、血管内皮细胞最为丰富。结论：层粘连蛋白受体一般见于牙髓复牙本质复合体的各种细胞 膜上，它与层粘连蛋白一起在支持、固定牙髓牙本质复合体各结构的正常位置、形态和功能的发挥上可能起了重要作用。     

机械性刺激对大鼠牙髓中P物质神经纤维表达的影响

目的：观察大鼠牙齿硬组织在受到不同程度机械损伤刺激时,牙髓中P物质神经纤维的表达,探讨机械性损伤刺激与神经肽P物质的关系。方法：采用间接免疫荧光法观察大鼠牙齿硬组织损伤时牙髓P物质神经纤维的表达情况。结果：随着机械性损伤程度加深,牙髓中P物质神经纤维末梢膨大、数量发生变化（P〈0.05）。结论：牙齿在受到不同程度机械性损伤刺激时,牙髓内P物质神经纤维表达发生变化。     

人前磨牙牙髓牙本质复合体层粘连蛋白受体的免疫组织化学研究

目的研究层粘连蛋白受体在牙髓牙本质复合体内的表达,探讨其分布与功能的关系.方法收集新鲜健康人前磨牙60个,固定,脱钙,石蜡包埋,层粘连蛋白受体SABC免疫组织化学染色.结果层粘连蛋白受体免疫反应物(LNR-IR)普遍存在于牙髓牙本质复合体的神经纤维膜、血管内皮细胞、成牙本质细胞及成纤维细胞上,以牙髓的神经纤维膜、血管内皮细胞最为丰富.结论层粘连蛋白受体一般见于牙髓牙本质复合体的各种细胞膜上,它与层粘连蛋白一起在支持、固定牙髓牙本质复合体各结构的正常位置、形态和功能的发挥上可能起了重要作用.     

LIM矿化蛋白1在大鼠牙髓损伤修复中的时空表达

目的:探讨LIM矿化蛋白1(LIM mineralization protein 1,LMP-1)在大鼠磨牙牙髓损伤修复中的表达.方法:建立大鼠磨牙牙髓损伤修复动物模型,用免疫组织化学染色方法观察LMP-1在大鼠牙髓损伤修复中的表达,并用Image-Pro Plus 6.0图像分析软件及单因素方差分析比较各组间的差异.结果:在正常大鼠牙髓组织中LMP-1表达阴性;在大鼠牙髓损伤后1d,LMP-1表达于成牙本质细胞及部分牙髓成纤维细胞;术后3 d,LMP-1表达于坏死牙髓下方的细胞增殖层;术后7d,LMP-1显著表达于增殖活跃的牙髓细胞及部分成牙本质细胞中.结论:LMP-1在牙髓损伤修复过程中呈时空特异性表达,可能参与成牙本质细胞及牙髓细胞的增殖、分化及修复性牙本质的形成.     

牛血浆纤维粘连蛋白对培养的人牙髓细胞ConA、WGA、SBA凝集素受体表达的影响

本文观察了不同浓度牛血浆纤维粘连蛋白（ＦＮ）和人工合成肽（ＲＧＤＳ．４０μｇ／ｍｌ）对人牙髓成纤维细胞ＣｏｎＡ、ＷＧＡ、ＳＢＡ凝集素受体的影响，培养液中ＦＮ的浓度分别为１０、２０、４０、８０μｇ／ｍｌ，ＲＧＤＳ的浓度为４０μｇ／ｍｌ．凝集素受体的表达情况通过图象分析仪进行半定量分析，培养的人牙髓细胞不表达ＳＢＡ受体，ＦＮ（４０、８０μｇ／ｍｌ）可显著地使人牙髓细胞ＣｏｎＡ受体表达增强，而ＷＧＡ的受体表达减弱，ＲＧＤＳ（４０μｇ／ｍｌ）组的细胞ＷＧＡ的受体表达弱于ＦＮ（１０、２０μｇ／ｍｌ）组。     

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目的探讨急性刺激对预先制备的大鼠受损牙齿牙髓的影响。方法本研究于2011年10-12月在大连大学附属新华医院实验中心进行。将24只sD大鼠的两侧上颌第一磨牙用涡轮机钻洞至牙本质深层,用氧化锌暂封,随机分为2组。应激组（12只）给予冷刺激,断食,断水;对照组（12只）正常饲养。在应激刺激第5、13、20、28天各组分别随机抽取3只大鼠,处死后取上颌双侧第一磨牙,检测牙髓炎症的变化72．P2X3受体表达情况。结果HE染色显示应激组大鼠牙髓充血水肿,有淋巴细胞、中性粒细胞等炎细胞浸润,后期出现牙髓细胞变性坏死。应激组牙髓中P2X3受体阳性表达在刺激第5、13天轻微增高,第20天明显增高,第28天逐渐下降;与对照组比较,差异有统计学意义（P〈0．05）。结论急性刺激可造成大鼠受损牙齿的牙髓炎症反应,从而导致大鼠出现牙髓来源的疼痛。     

Glutamate control of pulpal blood flow in the incisor dental pulp of the rat

Glutamate is present in primary sensory afferents innervating the dental pulp and is known to exert vasoactive effects. The aims of this study were (i) to assess pulpal blood flow (PBF) after glutamate infusion in the dental pulp and (ii) to observe the distribution of glutamatergic nerve fibers expressing the vesicular transporters of glutamate (VGluT). The PBF was monitored with laser Doppler flowmetry before and after glutamate (0.5 M) infusion in the dental pulp vs. saline infusion. Immunochemistry for VGluT1, 2, and 3 was performed in addition to immunochemistry for the vascular and neuronal markers smooth‐muscle actin (SMA), isolectin B4 (IB4), and calcitonin gene‐related peptide (CGRP). Glutamate infusion resulted in a PBF increase that lasted for 60 s. Positive immunolabeling was observed for the three glutamate transporters, but was more pronounced for VGluT3. Moreover, VGluT3 immunoreactivity was observed within nerve fibers entering the dental pulp and terminating at the periphery and at the vicinity of odontoblasts. Also, VGluT3 was colocalized with the vascular marker SMA, and in some nerve fibers with IB4, but not with CGRP. This study provides support for a control of dental pulp microcirculation by neurons expressing VGluT3.     

凝血酶受体在人牙髓组织和细胞中表达的免疫组化研究

目的：探讨凝血酶受体在人正常、炎症牙髓组织及体外培养的牙髓成纤维细胞中的表达及其意义。方法：体外培养人牙贿成纤维细胞,收集正常和炎症牙髓组织,采用免疫组织化学方法观察凝血酶受体在牙髓组织和细胞中的表达和分布。结果：体外培养的牙髓成纤维细胞可见受体强阳性表达,正常及炎症牙髓组织中成牙本质细胞层、牙髓细胞、血管内皮细胞均呈广泛的阳性表达,炎症区淋巴细胞、单核细胞、中性粒细胞染色为强阳性。结论：人牙髓组     

炎症信号受体TLR4在大鼠正常牙髓和牙周组织中的分布及意义

目的:研究大鼠正常牙髓和牙周组织中炎症信号受体TLR4表达特点,探讨其在健康牙髓及牙周组织天然免疫防御中的可能作用.方法:采用石蜡及冰冻切片和免疫组化技术检测大鼠健康牙髓及牙周组织中TLR4的表达情况.结果:正常牙髓组织中成牙本质细胞、血管内皮细胞及参与先天免疫的巨噬细胞、树突状细胞、中性粒细胞等细胞TLR4免疫反应阳性,牙髓成纤维细胞不表达TLR4.TLR4表达于牙周结缔组织,可见与上皮层临近的固有层结缔组织强阳性染色.结论:TLR4可能参与了牙髓牙周组织的先天免疫反应,在牙髓牙周组织抵御外来病原微生物入侵中可能起重要作用.     

Pressure induces interleukin-6 expression via the P2Y6 receptor in human dental pulp cells

Background and objective

An increase in intrapulpal pressure occurs during inflammation and restorative procedures; however, the role of the pressure on human dental pulp cell (HDPC) is not yet clarified. In this study, the effect of pressure on interleukin-6 (IL-6) expression of HDPCs was examined.

Design

HDPCs were applied with pressure (0.7–1.4 g/cm²). The level of IL-6 mRNA and protein release was determined by RT-PCR and ELISA, respectively. The signalling pathways were investigated using inhibitors, antagonists, and small interfering RNA.

Results

The results showed that pressure up-regulated IL-6 mRNA expression and protein release in a time- and dose-dependent manner. The implication of P2Y receptor was exhibited by a significant inhibition of pressure-induced IL-6 expression by suramin, an antagonist for the non-specific purinergic receptor family. Using loss of function experiments, we showed MRS2578 (a specific P2Y6 antagonist), as well as P2Y6 small interfering RNA, abolished pressure-induced IL-6, whilst MRS2179 (a specific P2Y1 antagonist) and NF449 (a P2X1, P2X3, P2Y1, and P2Y2 antagonist) had no effect. Finally, we demonstrated that either the conditioned medium collected from pressurised dental pulp cells or addition of UDP, a selective agonist of P2Y6, up-regulated IL-6 expression in HDPCs.

Conclusions

These results indicate that pressure could induce IL-6 expression through the P2Y6 receptor in HDPCs, leading to a new insight of the role of pressure on cytokine release during pulpal inflammatory process.     

Changes of CD90 expression and immunoreactive cell localisation in rat dental pulp after cavity preparation

CD90 expression and immunoreactive cell localisation in rat dental pulp cells after cavity preparation was investigated. Cavity preparation was performed on the maxillary first molar of 8‐week‐old Wistar rats (n = 36), and immunohistochemistry and quantitative real‐time PCR were performed. CD90‐immunoreactivity was observed among subodontoblastic cells in the control group. One day after cavity preparation, the CD90‐immunoreactivity disappeared under the cavity area. While CD90‐immunoreactivity was faint after 3 days, the re‐arrangement of odontoblasts was detected in contact with dentine. After 5 days, the odontoblasts were observed beneath the dentine, and CD90‐immunoreactive cells were localised under the odontoblast layer. Immunofluorescence showed co‐localisation of CD90 and nestin was detected after 3 days. After 5 days, CD90‐immunoreactivity increased at the subodontoblastic layer. mRNA expression of CD90 and DSPP decreased after cavity preparation, and gradually recovered (P < 0.01). These results suggest that CD90‐immunoreactive cells in the subodontoblastic layer contribute to regeneration of odontoblast and subodontoblastic layers following cavity preparation.     

Proliferation of dental pulp fibroblasts in response to thrombin involves mitogen-activated protein kinase signalling

AIM: To examine the involvement of mitogen-activated protein kinases (MAPK) signalling on thrombin-stimulated human dental pulp fibroblasts (DPF). METHODOLOGY: Dental pulp fibroblasts were isolated from dental pulp connective tissue of third molars and expanded in vitro. Expression of thrombin receptors was analysed by RT-PCR, and cell proliferation was measured by 3[H]-thymidine incorporation assay. Phosphorylation levels of MAPK were determined by Western blot analysis, and alkaline phosphatase activity was measured to serve as a marker for odontogenic differentiation. Statistical analysis was performed by Student's t-test. RESULTS: Dental pulp fibroblasts express the thrombin receptors protease-activated receptor-1 (PAR-1), PAR-3 and PAR-4. Measurement of 3[H]-thymidine incorporation revealed a dose-dependent increase of DNA synthesis in response to thrombin treatment. The thrombin-induced mitogenic activity was decreased by the extracellular signal-regulated protein kinase (ERK) signalling inhibitor PD98059 (P < 0.05), and by SB203580 (P < 0.05), a p38 MAPK inhibitor. Western blot analysis demonstrated increased phosphorylation of ERK in DPF following stimulation with thrombin, while p38 MAPK and c-Jun NH2-terminal kinase (JNK) were not activated. Alkaline phosphatase activity of DPF remained unchanged upon incubation with thrombin. CONCLUSIONS: These results suggest that signalling via MAPK mediates the mitogenic activity of thrombin on DPF and may thus play a role during the early stages of pulp repair.     

Neuropeptide Y Y1 receptor in human dental pulp cells of noncarious and carious teeth

Aim To determine the distribution of the NPY Y1 receptor in carious and noncarious human dental pulp tissue using immunohistochemistry. A subsidiary aim was to confirm the presence of the NPY Y1 protein product in membrane fractions of dental pulp tissue from carious and noncarious teeth using western blotting. Methodology Twenty two dental pulp samples were collected from carious and noncarious extracted teeth. Ten samples were processed for immunohistochemistry using a specific antibody to the NPY Y1 receptor. Twelve samples were used to obtain membrane extracts which were electrophoresed, blotted onto nitrocellulose and probed with NPY Y1 receptor antibody. Kruskal–Wallis one‐way analysis of variance was employed to test for overall statistical differences between NPY Y1 levels in noncarious, moderately carious and grossly carious teeth. Results Neuropeptide Y Y1 receptor immunoreactivity was detected on the walls of blood vessels in pulp tissue from noncarious teeth. In carious teeth NPY Y1 immunoreactvity was observed on nerve fibres, blood vessels and inflammatory cells. Western blotting indicated the presence and confirmed the variability of NPY Y1 receptor protein expression in solubilised membrane preparations of human dental pulp tissue from carious and noncarious teeth. Conclusions Neuropeptide Y Y1 is expressed in human dental pulp tissue with evidence of increased expression in carious compared with noncarious teeth, suggesting a role for NPY Y1 in modulation of caries induced pulpal inflammation.