Bmi-1缺失导致小鼠牙和下颌骨发育障碍

目的 探讨Bmi-1基因在小鼠牙齿和下颌骨发育中的作用.方法 利用利用影像学、组织学和组织化学及图像分析方法比较分析了2～4周龄同窝野生型和Bmi-1基因敲除小鼠牙齿和下颌骨的表型差异.结果 与同窝野生型小鼠相比,Bmi-1基因敲除小鼠牙齿和下颌骨矿化明显降低;牙量和下颌骨骨量也明显降低;成骨细胞数和碱性磷酸酶阳性面积明显降低,而TRAP阳性破骨细胞数无明显变化.结论 Bmi-1基因缺失引起小鼠牙形成和成骨细胞骨形成降低导致小鼠下颌骨牙量和骨量降低.因此,Bmi-1具有促进牙和下颌骨发育的作用.     

脑源性神经营养因子在大鼠炎性牙髓中的表达及意义

目的：观察脑源性神经营养因子（brain-derived neurotrophic factor BDNF）在大鼠炎性牙髓中的表达及其变化,初步探讨BDNF在牙髓损伤修复中的作用。方法：通过开髓建立大鼠牙髓炎模型,用免疫组化法及图像分析技术检测正常牙髓及牙髓炎1d组、3d组、5d组、7d组牙髓中BDNF的表达。结果：免疫组化显示正常牙髓组织中无BDNF表达,在炎症1d组弱表达,3d组时到达高峰,到第5天开始减弱,总体差异有统计学意义（P〈0.05）。结论：与正常牙髓组织相比,炎症牙髓组织中BDNF开始表达,提示BDNF在牙髓炎发展过程中起一定作用。     

Notch信号分子于小鼠牙髓干细胞样细胞表达的研究

目的　研究Notch基因在小鼠牙髓干细胞样细胞表达。方法　采用酶消化培养法获得小鼠的单个牙髓细胞悬液,调整细胞密度为1×104个/孔细胞,干细胞培养液培养14 d,挑选细胞克隆扩增,提取细胞的总RNA,反转录聚合酶联反应(RT-PCR)检测Notch基因的表达。结果　小鼠牙髓细胞呈集落状生长,克隆形成率约为1·6 ~2·5 个/104细胞,所形成的集落细胞结合紧密,细胞胞体小、胞核大,RT-PCR结果显示Notch的mRNA在牙髓干细胞样细胞中有表达。结论　培养的集落状生长的小鼠牙髓细胞具有干细胞增殖快的特性,Notch基因于牙髓干细胞中表达,表明Notch信号参与了牙髓干细胞样细胞的早期分化的调控过程。     

促红细胞生成素对人牙髓细胞增殖和成骨分化的影响

目的：探讨促红细胞生成素（EPO）对体外培养的人牙髓细胞（hDPCs）增殖和成骨分化的影响。方法：体外培养鉴定人牙髓细胞。使用 EPO 对在成骨诱导培养基中培养的牙髓细胞进行刺激,CCK-8检测 EPO 对牙髓细胞增殖的影响;20 U ／ml EPO 培养 hDPCs 7 d 和14 d,采用碱性磷酸酶活性（ALP）检测和茜素红染色观察 EPO 对牙髓细胞矿化的影响;利用 Real-time PCR 检测加入 EPO 后牙髓细胞成牙本质向分化相关基因的表达情况。结果：EPO 以时间和剂量依赖性方式促进牙髓细胞的增殖;20 U ／ml 的 EPO 后作用,牙髓细胞碱性磷酸酶活性显著提高（P ＜0．05）,钙结节形成明显增多;成牙本质向分化相关基因 DSPP、OCN、OSTERIX、RUNX2的表达明显上调（P ＜0．05）。结论：EPO 能促进人牙髓细胞的增殖和分化。     

p53 基因敲除 PLAG1 转基因小鼠模型构建的研究

目的：建立p53基因敲除plagl转基因小鼠模型。方法：依托plagl转基因多形性腺瘤小鼠。取两只plagl＋母鼠与一只p53＋／-雄鼠杂交,分别取F1代中基因型为plagl＋p53＋／-的雌雄小鼠交配,得到基因型分别为plagl＋p53-／-,plagl＋p53＋／-及plagl＋p53＋／＋的三种多形性腺瘤小鼠,分别测量比较三种基因型多形性腺瘤的生长速度。结果：plagl＋p53-／-基因型小鼠生长迟缓,寿命短,未能长出肿瘤便已死去。plagl＋p53＋／-肿瘤生长速度较同龄plagl＋p53＋／＋小鼠相比较快,且有明显的差异（P〈O．05）。结论：构建了p53基因敲除plagl转基因小鼠模型,且结果显示p53基因与PLAGl基因的相互作用和多形性腺瘤的生长速度增快有关。     

小鼠舌肌发育相关基因的功能聚类分析

目的：研究小鼠舌肌发育的分子调控机制。方法：取胚胎第13．25天（E13．25）及 E15．5小鼠舌组织。应用 Affy-metrix Mouse GeneChip,对胎鼠舌发育过程中的差异基因进行筛选。应用 DAVID 网络分析工具对基因进行功能和聚类分析。结果：基因功能和聚类分析表明,在 E13．25高表达的基因主要与细胞周期相关因子（Exo1、Gsk3B、Kif20b、Skp2）和细胞粘附因子（Neo1、lama1）等相关。在 E15．5高表达的基因主要与细胞骨架（titin、Hspb7）相关。结论：小鼠舌组织增殖和特化与细胞周期和细胞粘附基因相关,舌组织分化和成熟主要与细胞骨架相关。     

10％纳米羟基磷灰石聚醚醚酮浸提液培养成骨细胞（MG63）Wnt3a／β－catenin信号通路的表达

目的：研究10％纳米羟基磷灰石聚醚醚酮（10％ HA／SPEEK／PEEK）复合材料浸提液培养MG63细胞后Wnt3a／β－catenin信号通路中特征性蛋白的变化,评价复合材料的生物相容性。方法：Wnt3a持续作用于10％HA／SPEEK／PEEK浸提液培养的MG63细胞。应用免疫荧光染色技术,定性检测Wnt信号通路中β－catenin蛋白的存在。应用Western－Blotting技术相对定量检测β－catenin蛋白的多少。应用实时定量PCR技术相对定量检测信号通路下游基因c－myc、cyclinD1的表达情况。结果：免疫荧光染色和Western－Blotting实验结果显示,Wnt3a作用于10％HA／SPEEK／PEEK浸提液中培养的MG63细胞,β－catenin蛋白绿色深染,位于细胞核中,表达量显著增加。实时定量RT－PCR实验结果显示Wnt3 a作用于10％HA／SPEEK／PEEK浸提液中培养的MG63细胞,信号通路的下游靶向基因c－myc、cyclinD1表达显著增加,Wnt通路开放。结论：10％HA／SPEEK／PEEK对MG63细胞有良好的成骨作用,可以指导临床实验研究。     

MyomiRs 在维甲酸诱导舌发育异常中对舌肌分化调控的机制研究

目的：检测维甲酸（Retinoic acid,RA）诱导小鼠胚胎腭裂模型中胎鼠舌体发育过程中肌相关 microRNAs（MyomiRs）、成肌调节因子（Myogenic regulatory factors,MRFs）以及 Pax 基因的表达变化,探究 MyomiRs 在舌肌分化过程中的调控作用,推测RA 致胎鼠腭裂伴发舌异常的可能机制。方法：建立 RA 诱导小鼠胚胎腭裂模型,分别在 E13．5、E14．5、E15．5收集胎鼠舌体组织,用 SYBR GreenⅠ实时定量 PCR 检测舌体中 MRFs 和 Pax 基因的表达;用 TaqMan 探针实时定量 PCR 检测舌体中 MyomiRs 的表达。结果：胎鼠舌体发育过程中,正常组 miR-1和 miR-206相对表达量均持续上升,RA 诱导组二者变化趋势与正常组相似,但相对表达量均低于正常组,miR-1的结果在 E14．5和 E15．5具有统计学意义（P ＜0．01）,miR-206的结果在 E13．5具有统计学意义（P ＜0．05）。正常组和 RA 诱导组胎鼠舌体中 MyoD 和 Myf5相对表达量都在 E14．5达到峰值,随后下降。RA 诱导组 MyoD 的表达在 E14．5显著低于正常组（P ＜0．05）,在 E15．5显著高于正常组（P ＜0．01）;RA 诱导组 Myf5的表达在 E15．5显著低于正常组（P ＜0．05）。正常组和 RA 诱导组胎鼠舌体中 Pax3表达均在 E14．5达到峰值,Pax7表达均在 E15．5达到峰值。RA 诱导组 Pax3的表达在 E14．5显著高于正常组（P ＜0．05）;Pax7的表达则在 E13．5显著高于正常组（P ＜0．01）。结论：在舌肌分化过程以及RA 诱导腭裂胎鼠的舌发育异常中,miR-1／miR-206与 Pax3／Pax7及 Myf5／MyoD 的表达趋势具有相关性。RA 可能通过下调 miR-1／miR-206而靶向上调 Pax3／Pax7,进而下调 MyoD ／Myf5表达,从而抑制舌肌分化,导致舌肌发育异常。     

大鼠正畸牙移动中牙周组织Wnt10b和β-catenin的表达研究

［摘要］目的：通过观察Wnt10b和β-catenin在大鼠正畸牙齿移动牙周组织中的表达,初步探讨Wnt10b和β-catenin在正畸牙齿移动牙周组织改建中的作用。方法：建立30只雄性SD大鼠左侧上颌第一磨牙近中移动模型。右侧上颌第一磨牙不加力作为自身对照。分别在牙齿移动1d、3d、5d、7d、10d、14d时处死动物,制取上颌标本。进行免疫组织化学分析。结果：对照组大鼠牙周组织中,Wnt10b和β-catenin低表达。实验组牙周组织中,β-catenin表达增加在各时间点均有显著差异,Wnt10b仅在5d、7d、10d时有显著差异。结论：Wnt10b和β-catenin参与了正畸牙齿移动中牙周组织改建。     

Wnt通路抑制剂XAV-939对牙髓干细胞成脂分化的影响

目的：通过体外实验探讨Wnt通路抑制剂XAV?939对牙髓干细胞增殖及成脂分化的影响。方法酶消化法培养牙髓干细胞,鉴定后采用CCK8试剂盒检测XAV?939对牙髓干细胞增殖的影响,油红O染色检测XAV?939对牙髓干细胞成脂分化的影响,qRT?PCR检测成脂分化过程中,XAV?939对Wnt通路相关基因糖原合成酶激酶3β（glycogen synthase kinase?3β, GSK3β）和β?catenin以及成脂分化相关基因过氧化物酶体增殖物激活受体γ2（peroxisome proliferator activated receptor?γ2, PPARγ2）和CCAAT增强子结合蛋白α（CCAAT/enhancer binding protein α, C/EBPα）的影响。结果牙髓干细胞成脂诱导14 d后,XAV?939上调GSK3β、PPARγ2和C/EBPα的表达,同时下调β?catenin的表达。结论 XAV?939可以通过抑制Wnt通路促进牙髓干细胞的成脂分化。     

Mouse tooth development time sequence determination for the ICR/Jcl strain

To establish the normal dental development pattern of the ICR/Jcl strain of mouse, we analyzed a significant number of observations of the different developmental stages of the first mandibular molar, accurately recording the chronology of their daily embryonic development. Proliferation of the dental sheet began at day 12.5 in utero (E-12.5), the bud stage appeared at days E-13.5 and E-14.5, the cap stage was observed at days E-14.5, E-15.5 and E-16.5 and the early bell stage at day E-17.5. The presence of predentin was observed at day E-18.5 and dentin was observed 1 and 2 days after birth (D-1 and D-2). The late bell stage with presence of enamel was detected more than 3 days after birth. Embryonic and dental development in the ICR/Jcl strain of mouse is faster than in other well-known strains. The establishment of this developmental pattern will be useful for future investigations of transgenic mice.     

The role of canonical Wnt signaling in dentin bridge formation

ObjectiveWnt signaling has been reported to be involved in dentin bridge formation. However, the detailed mechanism has not yet been clarified. We elucidated the localization of canonical Wnt signaling molecules during dentin bridge formation.MethodsPulp of the maxillary first molar in mice was exposed and directly capped with MTA cement. Maxillae were collected on the 1st, 4th, 7th, 14th, and 28th days after treatment. After μCT analysis, immunohistochemistry for Wnt3a, Wnt10a, β-catenin, F4/80, and osterix was performed in paraffin-embedded sections.ResultsOn the 4th and 7th days after pulp capping, odontoblasts and dental pulp cells expressed Wnt3a, Wnt10a, and β-catenin. On the 14th day, reactionary dentin was formed around the pulp exposure area. Odontoblasts and dental pulp cells express Wnt3a, Wnt10a, and β-catenin. Additionally, F4/80- and Wnt10a-positive macrophages were observed at the center of the dental pulp. When the dentin bridge was formed on the 28th day, reparative odontoblasts expressed Wnt3a, β-catenin and osterix.ConclusionWnt ligands derived from odontoblasts and dental pulp cells are important for the activation of odontoblasts and the differentiation of reparative odontoblasts during dentin bridge formation. Macrophage-derived Wnts are also involved in reparative odontoblast differentiation.     

TGF-β3和牙髓干细胞修复损伤面神经效果的评价

目的：探讨在TGF-β3的诱导下牙髓干细胞对修复受损面神经的可行性,比较不同时间点面神经的恢复情况。方法：选择新西兰大白兔制作动物模型,人为制备兔损伤面神经标本,选择同体面神经标本作对照,随机分为1月组和3月组两个大组,通过牙髓干细胞和TGF-β3再生室进行修复,收集兔断端面神经标本,进行行为学观察、组织学观察分析,应用实时荧光定量PCR技术检测神经生长的特异性标志物S100和Nestin的基因表达水平。结果：1）行为学观察：实验组与对照组相比较,实验组再生神经恢复良好,损伤症状明显缓解。2）HE染色：实验组与对照组相比较,新生的神经纤维较多,神经束大小不均,束间血管多,神经纤维髓鞘化程度高,且厚度增大。3）实验组面神经中S100和Nestin均有特异性表达,其中S100在1月组mRNA的基因表达水平较3月组mRNA基因表达水平高,差异具有统计学意义（P=0.000）;Nestin在3个月组mRNA的基因表达水平较1个月组mRNA的基因表达水平高,差异具有统计学意义（P=0.001）。结论：通过行为学、组织学观察和荧光定量PCR技术检测神经生长特异性标志物S100和Nestin在mRNA基因水平的表达,证明通过TGF-β3的诱导作用,牙髓干细胞可定向分化成神经干细胞,从而起到修复面神经的作用。     

Effect of ageing on responses of nerve fibres to pulpal inflammation in rat molars analysed by quantitative immunocytochemistry.

The response of sensory nerve fibres to inflammation in young adult rat molars has recently been shown to include increases in nerve sprouting and neuropeptide content. The objective was to evaluate neural responses to class V dental preparations in molars of old (1-2 yr) as compared with young adult rats (3-4 months). Tissues were investigated immunocytochemically 4 days post-injury for the sensory neuropeptides calcitonin gene-related peptide (CGRP) and substance P. Quantitative image analysis of the material demonstrated that more immunoreactivity was present for CGRP than for substance P in intact control teeth for each age group. Four days after injury, both immunoreactivities were increased in pulp adjacent to the injury in both young and old teeth. The increase depended on at least three factors: (1) enhanced immunoreactivity of the nerve fibres; (2) increased terminal nerve sprouts near the injury and (3) elevated peptide content of the pulp tissue. Although the incidence of CGRP- and substance P-immunoreactive nerve fibres had decreased in older teeth, the proportional increases in both neuropeptides near the injury were greater in old than in young teeth, owing to a reduction in pulpal volume during ageing. Pulpal tissue was also immunostained for the low-affinity nerve growth factor receptor (p75-NGFR) as an index of pulpal ageing; and an extensive decrease was found in the old adult as compared to young adult rats. These results indicate that old rats maintain the capacity for nerve sprouting despite the decreases in p75-NGFR labelling of pulp cells, pulp volume and nerve fibre numbers that occur as part of dental ageing.     

还原型谷胱甘肽稳态在腭裂发生中的作用

目的： 观察还原型谷胱甘肽稳态的改变对腭中嵴上皮细胞形态及腭裂发生率的影响,探讨氧化自由基在四氯二苯二噁英（TCDD）致畸过程中的作用。方法： 将GD10的SPF级C57BL/6J孕鼠随机分为4组,TCDD组：腹腔注射生理盐水和TCDD灌胃;TCDD+丁硫氨酸-亚砜胺(BSO)组：腹腔注射BSO 4 h后给予TCDD灌胃;BSO组：腹腔注射BSO和玉米油灌胃;对照组：腹腔注射生理盐水和玉米油灌胃。光学显微镜和电子扫描电镜下观察胎鼠腭胚突的发育及细胞表面形态。统计各组小鼠在GD17的腭裂发生率。免疫组织化学方法检测各组腭中嵴上皮各时期CYP1A1蛋白的表达。采用SPSS13.0软件包对数据进行统计分析。结果： TCDD成功构建高致畸率的腭裂模型。BSO增加了5%的腭裂发生率,但是相对于TCDD组无显著差异(P>0.05)。TCDD组腭胚突上抬时间较对照组推迟1 d,添加BSO的TCDD组腭胚突上抬过程较正常组减慢。电镜下胚胎发育的各发育时期,腭中嵴上皮表面TCDD组与对照组有显著差异。免疫组织化学检测发现,TCDD组腭中嵴上皮可见CYP1A1高表达。结论： 扰乱体内还原型谷胱甘肽稳态,可能会减少对小鼠体内氧化自由基的消耗,从而影响腭胚突的融合,继而加速了TCDD的毒性,影响腭裂发生。     

碱性成纤维因子用于犬牙盖髓剂的实验研究

目的探讨碱性成纤维因子(bFGF)对犬牙髓损伤修复的影响。方法选取健康英国小猎兔犬牙齿64颗,分为bFGF盖髓组、Dycal盖髓组及ZOE盖髓组。进行直接盖髓术,观察术后14天和28天修复性牙本质形成及牙髓组织学变化。免疫组化检测骨形成蛋白表达情况。结果术后14天:实验组及阳性对照组有纤维性基质形成,无牙本质桥形成;术后28天:实验组及阳性对照组有骨样牙本质桥、管状牙本质形成,阴性对照组无牙本质桥形成。结论bFGF在体内能够有效诱导牙髓细胞分化,形成修复性牙本质。     

地塞米松通过Wnt/β-catenin信号通路诱导腭裂的实验研究

目的探讨地塞米松对腭发育关键时期腭突间充质细胞和上皮细胞增殖和凋亡的影响,以及Wnt/β-catenin信号通路的分子关联作用。 方法将80只怀孕8.5 d（E8.5）的C57孕母鼠平分为两组,地塞米松组行腹腔注射地塞米松（8 mg·kg^-1·d^-1）,对照组注射等量0.9%氯化钠溶液,持续至E12.5,分别取E13.5、E14.5、E15.5和E17.5的胎鼠头部制成石蜡切片,苏木精-伊红染色观察腭突的形态,BrdU和荧光TUNEL染色分别检测腭突细胞的增殖和凋亡情况,Western blot检测腭突细胞的Wnt/β-catenin信号通路活性。χ²检验分析组间细胞增殖的差异,t检验分析组间细胞凋亡的差异。 结果在E13.5阶段,对照组前部腭突间充质细胞增殖率为（40.1 ± 7.4）%,地塞米松组增殖率为（35.5 ± 8.2）%,差异无统计学意义（χ²= 3.16,P= 0.075）。在E14.5阶段,对照组前部腭突间充质细胞增殖率为（50.3 ± 10.0）%,地塞米松组增殖率为（32.9 ± 8.8）%,差异有统计学意义（χ²= 5.229,P= 0.011）。在E15.5阶段,对照组前部腭突间充质细胞增殖率为（31.3 ± 6.5）%,地塞米松组增殖率为（18.5 ± 5.7）%,差异有统计学意义（χ²= 4.433,P= 0.02）。但各时间点后部腭突间充质细胞和上皮细胞的增殖差异均无统计学意义。地塞米松组腭突Wnt/β-catenin信号通路的活性显著下降。 结论地塞米松通过下调Wnt/β-catenin信号通路抑制腭突间充质细胞的增殖而导致腭裂发生。     

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.     

四氯二苯对二恶英和地塞米松诱导小鼠腭裂及转化生长因子-β3和受体活化样激酶5的表达

目的建立四氯二苯对二恶英（TCDD）和地塞米松（DEX）联合诱导C57BL/6J小鼠腭裂模型,并在腭发育关键时期检测转化生长因子-β3（TGF-β3）和受体活化样激酶5（Alk5）基因的表达,探讨TCDD和DEX联合诱导胎鼠腭裂与TGF-β3和Alk5的相关性。方法在小鼠GD10 ～GD12,实验组小鼠连续3 d胃饲TCDD和腹腔注射DEX,空白对照组不做处理,于GD17.5体视显微镜下检测各组腭裂发生率,并于GD13.5、GD14.5、GD15.5 分别剪取胎鼠腭突提取RNA,采用实时荧光定量聚合酶链反应检测TGF-β3和Alk5基因表达。结果采用TCDD和DEX联合致畸,可诱导C57BL/6J胎鼠形成100%腭裂,建立了一种稳定适合分子生物学研究的腭裂动物模型。GD13.5时TGF-β3和Alk5基因表达水平在实验组与空白对照组之间差异均无统计学意义（P>0.05）,在GD14.5、GD15.5实验组TGF-β3表达均降低（P<0.05）,而Alk5表达均升高（P<0.05）。结论TCDD和DEX联合作用可诱导C57BL/6J胎鼠形成稳定腭裂,在腭融合关键时期诱导TGF-β3表达下降,Alk5表达升高,与腭裂的发生具有一定的相关性。     

The effect of capsaicin on substance P expression in pulp tissue inflammation

AIM: To evaluate the effect of capsaicin on substance P (SP) expression during induced inflammation in rat pulp tissue. METHODOLOGY: Radioimmunoanalysis was used to measure SP levels in 36 mandibular molar pulps taken from six Wistar rats. Twelve samples were obtained from healthy pulps and used as negative control group. Another 12 samples were obtained after inducing inflammation with mechanical pulp exposure; these were used as the positive control group. Capsaicin was infiltrated into the inferior dental nerve in the experimental group and 12 samples were obtained after mechanical pulp exposure. RESULTS: The lowest SP expression was found in mechanically exposed pulps where capsaicin pretreatment had been carried out (0.028 ng mL(-1)), followed by healthy pulps (0.302 ng mL(-1)). The highest SP expression was found in mechanically exposed pulps with no capsaicin pretreatment (124 ng mL(-1)). The Kruskal-Wallis test showed statistically significant differences between the groups (P < 0.001). CONCLUSION: Inferior dental nerve infiltration with capsaicin reduces SP expression in dental pulp tissue in rats.