PHEX抗体对小鼠牙胚发育影响的实验研究

目的:研究PHEX抗体对小鼠牙胚发育的影响,探讨PHEX在牙齿发育过程的作用.方法:E17孕鼠尾静脉注射浓度分别为0.1mg/kg(A组)和0.5mg/kg(B组)的PHEX抗体.对照组N组注射0.1mol/LPBS.采用Mallory三色法染色观察钟状早期(E18)、钟状晚期(P1)、牙齿硬组织形成和矿化期(P3)牙胚的发育情况.免疫组织化学染色观察牙胚中PHEX蛋白的表达情况.结果:Mallory三色法染色结果显示;3组牙胚中釉质的发育无显著差异;成牙本质细胞的分化和牙本质的形成在A、N组中未见明显区别,而在B组中则明显延迟和抑制.E18、P1、P3 3组成釉细胞中的PHEX蛋白在牙胚中的表达均呈强阳性.P1、P3 A组和N组中成牙本质细胞中的PHEX表达也呈强阳性,但B组在P1期靠近基底膜的牙乳头细胞中表达呈弱阳性,在P3期也仅有少量的弱阳性表达.结论:0.5 mg/kg浓度的PHEX抗体可显著抑制成牙本质细胞的分化以及PHEX在其中的表达,进而抑制牙本质的生成;PHEX抗体对成釉细胞的发育和釉质的矿化无明显作用.     

小鼠牙胚发育过程中PHEX基因的时空表达模式研究

目的:研究PHEX在Balb/c小鼠牙胚发育过程中的时空表达模式,探讨其在牙齿发育过程中的作用.方法:采用免疫组织化学方法检测小鼠牙胚发育各时期中PHEX蛋白的表达.结果:PHEX在蕾状期无表达;帽状期的内、外釉上皮细胞和牙囊细胞中,PHEX有零星的弱阳性表达;钟状早期,PHEX主要表达在内釉上皮细胞和牙囊细胞,外釉上皮层、中间层细胞和靠近基底膜的牙乳头细胞中也有少量表达.钟状晚期,PHEX在成釉细胞和成牙本质细胞中的表达随着牙胚的发育而增强.釉结中成釉细胞的阳性表达程度比其他部位的成釉细胞更强.颈环处的内釉上皮细胞有PHEX的表达,而邻近的牙乳头细胞和外釉上皮细胞中无表达.结论:PHEX在牙胚的发育中有特定的时空表达模式,提示其参与了牙胚的形成发生和矿化.     

微管相关蛋白-4和β-微管蛋白在小鼠磨牙牙胚发育中表达的研究

目的:以小鼠磨牙为发育模型,研究其不同发育时期微管相关蛋白-4(microtubule-associated protein-4,MAP-4)和β-微管蛋白(β-tubulin)的表达特点,分析两者在牙齿发育中的作用。方法:取不同胎龄的胎鼠,制作其下颌第一磨牙切片,进行免疫荧光染色,荧光显微镜下观察MAP-4和β-tubulin的表达。结果:MAP-4和β-tubulin在牙胚蕾状、帽状、钟状期牙源性上皮和牙乳头内均有表达,随着发育的成熟表达逐渐增强,到硬组织形成期,二者的表达开始减弱。结论:MAP-4和β-tubulin参与了成釉器和牙乳头的发育和分化过程的调节,与牙胚细胞的增殖以及成釉细胞和成牙本质细胞的分化相关。     

基质金属蛋白酶-8在人和大鼠牙胚发育钟状期的表达

目的　研究基质金属蛋白酶-8(MMP-8)在牙胚发育牙本质形成过程中的表达。方法　免疫组织化学方法检测MMP-8在人牙胚发育钟状期的表达;原位杂交方法检测MMP-8 mRNA在大鼠牙胚发育钟状期的表达。结果　 MMP-8在人牙胚钟状晚期硬组织形成期成牙本质细胞和牙本质基质阳性表达,近髓腔处,牙本质染色更深;MMP-8 mRNA在大鼠牙胚发育钟状早期个别分化的成牙本质细胞表达阳性,钟状晚期,即硬组织形成期,MMP-8mRNA在成牙本质细胞表达强阳性。结论　MMP-8可能参与了人和大鼠牙胚发育过程牙本质基质改建。     

小鼠牙胚发育过程中collagenⅠ/Ⅲ的表达分布

目的:探讨collagenⅠ、Ⅲ在Balb/c小鼠牙胚发育各期的时间和空间分布特点,及两者在牙齿发育矿化中的作用。方法:用免疫组化方法检测小鼠牙胚发育各期collagenⅠ、Ⅲ的表达、分布。结果:collagenⅢ:出生前E13d小鼠牙胚蕾状期:口腔上皮细胞阳性表达( );E14~17d帽状期:口腔上皮细胞、成釉器星网层细胞表达弱阳性;E18~19d和出生后P1d钟状期与分化期:口腔上皮细胞、成釉器星网层细胞、牙乳头细胞、牙囊细胞表达阳性( );出生后P2~10d牙冠发育成熟期:成釉细胞、釉基质、成牙本质细胞、前期牙本质、牙乳头细胞、牙囊细胞、牙髓组织等均表达阳性( );P10~30d牙根发育成熟期:除上述细胞成分外,上皮根鞘、根周及根尖牙槽骨、牙骨质和牙周膜表达强阳性( )。collagenⅠ:在蕾状期无表达;帽状期:口腔上皮细胞、成釉器星网层细胞表达阳性;钟状期与分化期:口腔上皮细胞、成釉器星网层细胞、牙乳头细胞、牙囊细胞表达阳性;牙冠和牙根发育期:其分布和表达类似于collagenⅢ。结论:鉴于collagenⅠ、Ⅲ的表达和分布,表明它们均参与了牙胚和牙体硬组织的发育形成,但似乎cllagenⅢ的作用比collagenⅠ更为广泛。     

核心结合因子a1在小鼠牙齿发育过程中的表达

目的：观察Cbfal蛋白在小鼠牙胚发育过程中的表达情况。方法：用自制的Cbfal多克隆抗体，采用免疫组化染色观察其时空表达特性。结果：Cbfal蛋白在帽状期牙胚中表达较广泛，在钟状早、中期牙胚表达于内釉细胞、成釉细胞以及紧靠成牙本质细胞层的牙乳头细胞，而在钟状晚期，Cbfal在牙乳头表达为阴性，成牙本质细胞层弱阳性，成釉细胞为阳性或强阳性。结论：Cbfal可能在小鼠牙胚发育，尤其是在成牙本质细胞和成釉细胞分化过程中具有重要作用。     

氯离子通道CLC-7在小鼠牙胚中的表达

目的:研究氯离子通道CLC-7在小鼠牙胚发育过程中的表达及分布,探讨其在牙齿发育过程中的作用.方法:提取小鼠牙胚中的总RNA并制备不同发育阶段的小鼠牙胚标本,应用RT-PCR和免疫荧光染色方法检测CLC-7在小鼠牙胚中的表达及分布.结果:RT-PCR检测发现新生小鼠牙胚有Clcn7 mRNA的表达.免疫荧光染色发现E18小鼠牙胚开始有CLC-7的表达,CLC-7存在于牙胚多种细胞,如内釉上皮层细胞、中间层细胞、星网状层细胞、牙乳头细胞、成釉细胞、成牙本质细胞.结论:CLC-7在小鼠牙胚中呈特异性空间分布,可能参与牙胚发育过程的调节.     

P75NGFR在小鼠牙胚发育中的定位分布研究

目的：研究神经生长因子受体P75NGFR在小鼠牙胚发育过程中的定位分布。方法：采用免疫组化方法和图像分析技术观察P75NGFR在小鼠牙胚发育各期的表达与分布。结果：P75NGFR在小鼠牙胚发育各期均有不同程度的表达。胚胎E13d牙胚位于蕾状期：口腔上皮、成釉器上皮细胞阳性表达（＋）；在帽状期及钟状期口腔上皮、成釉器的星网层表达阳性（＋）；在P2～10d牙冠发育成熟期：成釉细胞、成牙本质细胞、前期牙本质、牙乳头细胞、牙囊细胞表达阳性（＋）；P10—30d牙根发育期：除上述细胞成分外，上皮根鞘、牙槽骨表达强阳性（＋＋）。结论：P75NGFR参与牙胚及牙体硬组织的发育过程。     

Ddit3在小鼠下颌第一磨牙牙胚不同时期的表达研究

目的:检测Ddit3在小鼠下颌第一磨牙牙胚不同时期的表达分布及变化规律,初步揭示Ddit3在小鼠牙胚发育中的作用。方法:取不同胚胎时间点的ICR妊娠小鼠,制备牙胚发育标本,通过免疫荧光和免疫组化的方法显示Ddit3在小鼠下颌第一磨牙牙胚发育不同时间点的表达分布。结果:Ddit3在牙胚发育的早期主要表达于胞浆中,从钟状期开始,Ddit3不仅在胞浆中有阳性表达,同时也微弱地表达于细胞核中。分布如下:在小鼠下颌第一磨牙发育的板状期,Ddit3几乎没有表达。在蕾状期,Ddit3主要表达于釉上皮的细胞质中,在牙外胚间充质细胞中几乎无表达。在帽状期,Ddit3的表达模式基本和蕾状期相同。在钟状期早期,Ddit3在成釉细胞、前成牙本质细胞和牙乳头胞质中呈阳性表达,在部分细胞核中也检测到了Ddit3的表达。钟状期晚期,Ddit3在新形成的硬组织周围的高柱状成釉细胞、成牙本质细胞和牙乳头细胞的胞浆中有阳性表达,在大多数成釉细胞、成牙本质细胞和牙乳头细胞的细胞核中也有表达。结论:Ddit3可能会调节成釉细胞和成牙本质细胞的分化及其硬组织的生物矿化。     

ADAM28在小鼠牙胚发育中的时空表达

目的:研究ADAM28在小鼠牙胚发育中的时空分布。方法:采用免疫组织化学方法和图像分析技术观察ADAM28在小鼠牙胚发育各期的表达分布及差异。结果:ADAM28在牙胚发育各时期均有不同程度的表达。帽状期开始即在口腔上皮及成釉器星网层细胞、基底膜、牙乳头细胞和牙囊细胞表达强阳性,到钟状晚期,成釉细胞、釉基质、上皮根鞘和牙乳头细胞阳性表达;至冠根硬组织发育期,成釉细胞、成牙本质细胞、上皮根鞘、成牙骨质细胞、牙乳头细胞、牙囊细胞阳性表达。结论:ADAM28作为上皮和间充质间重要的信号分子,参与了从蕾状期到钟状晚期、从基质分泌到硬组织形成的牙冠、牙根形态发生过程,它可能在牙源性间充质细胞的早期形成、增殖与分化启动中发挥重要作用。     

Regulation of IGF-I by IGFBP3 and IGFBP5 during odontoblast differentiation in mice

ObjectivesAlthough intracellular signaling pathways of insulin-like growth factor I (IGF-I) related to the proliferation of dental pulp cells have been investigated, the switching mechanism from cell proliferation to differentiation during odontogenesis remains elusive. This study aimed to elucidate the role of IGF binding protein (IGFBP) 3 and 5 in regulation of IGF-I during odontoblast differentiation in mouse incisors.MethodsThe detailed expression patterns of IGF-I, IGF-I receptor (IGF-IR), IGFBP3, and IGFBP5 together with that of an odontoblast differentiation marker, nestin, were examined by immunohistochemistry and/or in situ hybridization using paraffinized sections of TetOP-H2B-GFP mouse incisors at postnatal 4 weeks.ResultsUndifferentiated dental papilla cells and preodontoblasts (preOB) showed intense IGF-I- and IGF-IRα-positive reactions, and the expression was observed in differentiated odontoblasts, such as immature odontoblasts (iOB) and mature odontoblasts (mOB). IGFBP3/Igfbp3 was transiently expressed in preOB and early iOB, and the intensity of expression gradually reduced with the progression of odontoblast differentiation. In contrast, immunohistochemical analysis for IGFBP5 identified a positive reaction in the undifferentiated dental papilla cells and differentiated odontoblasts, and the expression of Igfbp5 was reduced in the differentiated odontoblasts.ConclusionThe present study demonstrated the expression patterns of IGF-I, IGF-IR, IGFBP3, and IGFBP5 during odontoblast differentiation in mouse incisors. These results suggested that IGFBP3 regulates the transition from the proliferative to differentiation stage by inhibiting the action of IGF-I on the proliferation of dental papilla cells, and that IGFBP5 plays an important role in the maintenance of the differentiated odontoblasts during tooth development.     

同源盒基因Msx-1、Msx-2和Dlx-2在小鼠下颌第一磨牙发育阶段的表达

目的 观察同源盒基因Msx—1、Msx-2和Dlx-2 mRNA在小鼠下颌第一磨牙发育阶段的表达。方法 取胎龄E11～E18和新生P1～P3小鼠的头或下颌，制备5μm连续冠状切片。体外转录合成地高辛标记的Msx—1、Msx-2和Dlx-2 RNA探针。原位杂交后观察Msx—1、Msx—2和Dlx—2在小鼠下颌第一磨牙中的表达。结果 在牙胚发育过程中，Msx—1转录信号始终只在间质细胞中观察到，在上皮细胞中呈阴性。Msx-2和Dlx-2在牙源性上皮和间质中均表达，在磨牙发育起始期二者表达相似，但在随后的牙胚发育过程中，它们出现不同的表达特征。结论 牙胚发育过程中，Msx—1、Msx-2和Dlx-2有不同的表达特征，可能共同参与调控上皮和间质问的相互作用。     

牙本质磷蛋白在人牙胚发育中的原位杂交研究

目的：观察牙本质磷蛋白（DPP）基因在人牙胚组织发育过程中的表达，探讨其在牙齿发育中的作用。方法：采用原位杂交的方法，检测帽状期、钟状期牙胚组织DPPmRNA的表达。结果：人牙胚帽状期、钟状早期均未检测到DPPmRNA的表达，在钟状晚期前期牙本质形成时成牙本质细胞、前成釉细胞中呈阳性表达。结论：牙本质磷蛋白基因的转录具有明显的的时空特异性，它可能在牙体硬组织的形成中起重要的作用。     

Calbindin-D28 kappa localization in rat molars during odontogenesis

Specific antiserum raised against Calbindin-D28 kappa, a vitamin D-dependent calcium-binding protein (CaBP) isolated from chick intestine, was used for localization of the protein in developing rat molars. Previously, CaBP had been localized in specific cells associated with the continuously erupting rat incisor: late pre-secretory ameloblasts, secretory and maturation zone ameloblasts, stratum intermedium cells adjacent to ameloblasts in the late zone of enamel secretion, and papillary cells underlying maturation zone ameloblasts. In this study, the peroxidase anti-peroxidase technique was used for localization of the peroxidase anti-peroxidase technique was used for localization of CaBP in histological sections of rat mandibles from 18-day-old rat embryos through 20-day-old neonates. CaBP was not detected in any cells of the enamel organ, dental papilla, or dental sac during early odontogenesis from the dental lamina stage through the advanced bell stage. The protein first appeared in secretory ameloblasts which were situated opposite odontoblasts with newly secreted dentin. CaBP was present in the cytoplasm of more mature ameloblasts, but not in less mature ameloblasts. Some stratum intermedium cells subjacent to well-developed secretory and maturation zone ameloblasts also contained CaBP. The protein was not detected in odontoblasts, pulp cells, or other cells associated with the developing molars. It was also absent from the demineralized enamel and dentin matrix. In developing rat molars, the time-course of appearance of CaBP, a protein dependent for its synthesis on the vitamin D endocrine system in other organ systems, suggests a potential direct role of this hormonal system in enamel mineralization.     

组蛋白去甲基化酶JMJD3在小鼠牙发育中的表达

目的 研究组蛋白去甲基化酶JMJD3在牙发育过程中的表达.方法 采用免疫组织化学方法观察小鼠胚胎E14~18天及出生后P1~P3磨牙牙胚中组蛋白去甲基化酶JMJD3的表达.结果 JMJD3表达于细胞核内,包括上皮性细胞和间充质细胞.在小鼠磨牙牙胚发育的蕾状期(E14)和帽状期(E16),JMJD3在牙蕾上皮,成釉器内釉细胞、外釉细胞、星网状细胞中弱表达.在小鼠磨牙牙胚发育的钟状期(E18),JMJD3在成釉器的外釉细胞层,内釉细胞层,中间层、星网状层及邻近间充质细胞内呈强阳性表达.在小鼠磨牙牙胚发育钟状晚期(P3), JMJD3在成釉细胞、成牙本质细胞及邻近间充质细胞呈强阳性表达.结论 JMJD3在小鼠牙发育过程中呈时空特异性表达,提示JMJD3参与小鼠磨牙的发育.     

牙本质涎磷蛋白、Ⅰ型胶原蛋白在vps4b基因敲除鼠磨牙牙胚发育中的时空表达

目的 研究vps4b基因突变对牙齿发育相关蛋白——牙本质涎磷蛋白（DSPP）和Ⅰ型胶原蛋白（COL-Ⅰ）表达的影响。方法 取胚胎E13.5 d、E14.5 d、E16.5 d的胎鼠头部及出生后P2.5 d、P7 d的幼鼠下颌骨组织,石蜡包埋后获取第一磨牙牙胚组织切片,采用免疫组织化学染色法检测野生型小鼠和vps4b基因敲除小鼠牙胚中DSPP、COL-Ⅰ的表达。结果 野生鼠蕾状期和帽状期DSPP、COL-Ⅰ均未表达;钟状期DSPP在内釉上皮和牙乳头中有表达,COL-Ⅰ表达于牙乳头和牙囊;分泌期和矿化期DSPP、COL-Ⅰ在成釉细胞、成牙本质细胞、牙囊内均有表达,COL-Ⅰ在牙乳头内亦可见表达。小鼠vps4b基因敲除后,DSPP在钟状期牙乳头和分泌期牙乳头及牙囊内未见表达,COL-Ⅰ在钟状期及矿化期表达部位与野生型小鼠一致,分泌期在牙乳头的表达发生改变。结论 vps4b基因在牙胚发育中发挥重要作用;DSPP、COL-Ⅰ的表达可能受vps4b基因的调控,并与vps4b共同调节牙齿牙本质的发育。     

Regulation of Osteoblast and Odontoblast Differentiation by RUNX2

Runx2-deficient mice completely lack osteoblasts and bone formation. Overexpression of Runx2 in osteoblasts inhibits osteoblast maturation, leading to immature bone, which is easily resorbed, while the expression of dominant-negative Runx2 in osteoblasts increases the volume of trabecular bone by promoting the formation of mature bone, which is relatively resistant to bone resorption. Thus, RUNX2 directs mesenchymal stem cells to the osteoblast lineage, and supplies immature osteoblasts to form immature bone, but RUNX2 has to be down-regulated for terminal differentiation into mature osteoblasts, which form mature bone. Thus, the level of RUNX2 determines the maturational stage of osteoblasts, bone maturity, and the bone turnover rate. Endogenous RUNX2 protein is detected in the nuclei of preodontoblasts, immature odontoblasts, and mesenchymal cells in the dental sac at 3 days of age, and transiently detected in ameloblasts in the coronal regions at one week of age. Overexpression of Runx2 in odontoblasts inhibited their terminal differentiation. Further, dentin sialophosphoprotein (Dspp) expression was lost and nestin (NES) expression was markedly decreased, while the expressions of bone gamma -carboxyglutamate (gla) protein 1/osteocalcin (BGLAP), osteopontin (SPP1), and dentin matrix protein 1 (DMP1) were up-regulated in the odontoblasts, resulting in the formation of a bone structure. These findings indicate that RUNX2 is able to induce the transdifferentiation of odontoblasts into osteoblasts, and that RUNX2 expression has to be down-regulated during odontoblast differentiation to achieve full differentiation for dentinogenesis.