猪牙源性间充质与胶原类材料复合移植的实验研究

目的：探讨经体外培养的出生后的猪牙源性间充质细胞成牙能力。方法：将体外培养第4代的猪牙胚细胞接种到胶原凝胶基质中,体外培养3d后进行裸鼠体内移植,6W后取材观察。结果：猪牙胚细胞原代培养过程中可见上皮样细胞与成纤维样细胞混杂存在,传代3次后上皮样细胞消失。细胞胶原基质复合物取材结果显示细胞聚集的区域可见牙本质-牙髓复合体样结构。结论：经体外传代培养后未经体外诱导的牙胚细胞接种到胶原凝胶基质所构建的组织工程牙齿有牙本质-牙髓复合体样结构形成,说明牙胚细胞经体外培养后其所获得的发育信息仍可保持,是研究牙齿发育构建模型及组织工程牙齿的理想的种子细胞。     

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。     

出生后牙胚细胞肾被膜移植的成牙能力研究

目的：将出生后牙胚细胞接种于肾被膜下探讨其成牙能力。方法：通过酶消化法获得出生后4d SD仔鼠第一磨牙牙胚细胞，离心后与明胶海绵支架复合植入大鼠肾被膜下，2周后组织学观察移植物中的组织新生情况。结果：细胞与支架复合体形成了包含釉质样组织和牙本质牙髓复合体的牙齿样结构，形成的硬组织与正常牙齿的形态结构与排列方式相似。Masson’s三色法显示绿色矿化基质形成。结论：出生后牙胚细胞仍保留了牙齿发育的遗传信号，体内移植后牙源性上皮细胞与间充质细胞相互作用，能重新排列构建出与正常牙体组织类似的规则排列结构，在适当条件下具有形成组织工程牙齿的潜能。     

牙本质磷蛋白和成釉蛋白在组织工程牙胚的表达模式研究

目的：采用大鼠牙胚细胞与胶原蛋白构建组织工程牙齿,通过免疫荧光组织化学分析牙齿特异性蛋白在组织工程牙齿的表达模式。方法：采用出生后4dSD仔鼠磨牙牙胚细胞,培养后将牙胚细胞与胶原蛋白溶液混合构建组织工程牙胚,2W后取材,HE染色观察移植物中的成牙能力,免疫荧光组织化学染色观察牙本质磷蛋白DSP和成釉蛋白AM的表达模式。结果：组织工程牙胚移植后2W可在肾被膜下形成乳白色矿化组织,HE染色可见典型的牙髓牙本质复合体样组织和釉质样结构,免疫荧光组织化学染色观察可见成釉蛋白AM阳性的釉质样结构和牙本质磷蛋白DSP阳性的牙乳头样组织,与正常牙齿组织表达模式一致。结论：采用牙胚细胞可与胶原蛋白进行重新排列形成组织工程牙胚,本实验从组织和蛋白分析上证实了其具有较强的成牙能力,为以后的临床应用提供了理论基础。     

Notch1蛋白在小鼠下颌切牙发育中的表达

目的探讨Notch1在小鼠下颌切牙牙胚发育过程中的组织学分布。方法制作ICR小鼠下颌切牙不同发育阶段的冰冻组织切片,对小鼠下颌切牙牙胚自牙胚发育起始期至钟状晚期不同发育阶段组织Notch1的分布情况进行免疫组织化学染色。结果 Notch1在小鼠下颌切牙发育蕾状期牙胚的口腔侧上皮中表达,而在和间充质相邻的牙胚上皮中没有表达。从帽状期至钟状期,Notch1在牙胚的中间层表达,而在内釉上皮中没有表达。钟状期的唇侧颈环部位星网状层和部分牙上皮细胞也表达Notch1。此外,Notch1还在牙胚发育不同时期的间充质、牙乳头和早期牙髓中表达。结论 Notch1可能在小鼠下颌切牙发育过程中的牙上皮,特别是内釉上皮的细胞分化,以及牙囊和牙乳头细胞分化及分化完成后的牙髓干细胞的稳定性方面有重要作用。     

牙齿发育中程序性细胞死亡的研究

目的：阐明程序性细胞死亡在牙齿发生、发育过程中的意义。方法：采用原位末端标记法（ＴＵＮＥＬ法）及ＨＥ染色技术，对纯系ＳＤ大鼠牙胚发育不同阶段的程序性细胞死亡现象进行观察。结果：在牙齿发育的蕾状期、帽状期、钟状期均有程序性细胞死亡现象。蕾状期主要出现在上皮细胞增殖内陷形成的蕾状突起的顶端，帽状期、钟状早期集中出现在细胞增殖生长中心（原发性釉结节，颈环）处，钟状晚期及牙体组织形成后成釉器星网层及牙乳头细胞中多见，外釉上皮及已分化的成釉细胞、成牙本质细胞中散在。结论：在牙齿发育形成过程中，程序性细胞死亡参与上皮－间充质相互作用。控制着不同状态下细胞增生的数量，及时清除多余细胞，完成其对牙齿发育的重要塑形作用。     

用人牙源性上皮和间充质细胞构建组织工程化牙齿样结构的实验研究

目的：以人牙源性上皮和间充质细胞作为种子细胞在裸鼠体内构建牙齿样结构。方法：将体外培养的人牙源性上皮细胞和经生长因子诱导后的牙源性间充质细胞分层接种于经塑形的陶瓷化骨或Collagraft三维支架上，将形成的细胞/支架复合物移植到免疫缺陷小鼠的皮下，建立牙源性上皮细胞复合间充质细胞的体内立体培养模型。14～18周后取材，采用HE染色、改良丽春红三色法、免疫组化和原位杂交方法检测移植物中的组织新生情况。结果：细胞在三维支架上形成了包含釉质样组织和牙本质牙髓复合体的牙齿样结构。新生的釉质样组织表达人成釉蛋白，其周围残留的牙源性上皮细胞表达人釉原蛋白mRNA。同时，人DSP蛋白在牙本质样组织中呈阳性表达。所有对照组标本中均未观察到该现象。结论：以优选出并经塑形的三维支架为载体，在裸鼠体内建立牙源性上皮细胞复合间充质细胞的立体培养模型，成功构建出组织工程化牙齿样结构。     

人牙本质涎蛋白在人牙胚发育过程中的表达和意义

目的：探讨人牙本质涎蛋白(human dentin sialoprotein，hDSP)在人牙胚发育过程中的表达和意义。方法：用免疫组化方法检测人牙本质涎蛋白在人牙胚不同发育阶段中的表达。结果：hDSP在蕾状期、帽状期釉上皮以及钟状早期内釉上皮有弱阳性表达，钟状中期正在分泌基质的成牙本质细胞、牙本质小管有强阳性表达，钟状晚期，牙本质小管仍有强阳性表达，而成牙本质细胞转为弱阳性表达。前成釉细胞、成釉细胞有一过性表达。前期牙本质始终无阳性表达。牙胚周围骨组织、软骨组织和口腔软组织无阳性表达。结论：提示hDSP可能参与了牙本质的形成。另外前期牙本质阴性表达，表明hDSP蛋白由成牙本质细胞分泌后，可能通过成牙本质细胞突起穿过前期牙本质分泌至矿化前沿，参与牙本质的形成。     

Notch 1信号蛋白在大鼠牙胚发育中的免疫组化表达研究

目的通过检测大鼠牙胚发育不同阶段Notch1信号的表达情况，探讨其在牙齿发育过程中的作用。方法采用免疫组织化学染色技术观测大鼠孕期12、14、16、17、18、19d和出生后1、3、5、7、9d牙胚中Notch1的表达。结果Notch1的表达始于钟状中期。在钟状中晚期，Notch1的表达在牙乳头间充质、成牙本质细胞层呈弱阳性。在内釉细胞层呈阳性。牙体组织形成开始，Notch1则在成釉细胞的中间层有反复表达，在成牙本质细胞层中有一过性表达。牙齿萌出后，该信号在成牙本质细胞和成釉细胞表达均为阴性。结论Notch1在牙胚发育过程中与成釉细胞分化有关，它可能在牙齿发育早期基质触发前成釉细胞向成釉细胞分化中起重要作用。     

小鼠牙胚的鸡胚培养模型的建立

目的　采用鸡胚建立一种新的小鼠牙胚体内培养模型。方法　取15 d龄的昆明胎鼠下颌第一磨牙牙胚, 将其用钨丝固定于孵化4~5 d(翼芽期)的鸡胚内,继续孵化10 d后,终止培养并取材,进行苏木精-伊红染色,常规组织学观察。结果　小鼠牙胚在鸡胚内可继续生长发育,从帽状期进入到钟状末期,镜下可见分化成熟的成牙本质细胞、成釉细胞和分泌的基质。结论　以翼芽期鸡胚作为小鼠牙胚体内培养的宿主,可以保持其发育,再现牙齿形成过程中的细胞分化、形态发生的生理过程。     

GD3 synthase gene found expressed in dental epithelium and shown to regulate cell proliferation

GD3 synthase is one of the key enzymes involved with ganglioside synthesis, and its activity regulates the main profile of ganglioside expression. We analyzed the expression of the GD3 synthase gene in laser-dissected teeth germs using RT-PCR. The GD3 synthase gene was found expressed in brain, thymus, and tooth germ tissues, however, not in liver or skin specimens. Further, it was highly expressed during the early stage of tooth germ development (embryonic day 14.5), especially in dental epithelia, which gradually reduced in the molar site until postnatal day 7, whereas it was not in dental mesenchyme tissues. In addition, dental epithelial cells transiently transfected with the GD3 synthase gene showed enhanced proliferation. These results indicate that the GD3 synthase gene may be involved in early tooth development, particularly in the proliferation of dental epithelium.     

Apoptosis in regressive deciduous tooth germs of Suncus murinus evaluated by the the TUNEL method and electron microscopy

Apoptosis in regressive primary (deciduous) dental primordia was examined in the embryos of Suncus murinus, which is a monophyodont. The primary tooth germs of S. murinus are temporarily formed and disappear during the embryonic period before they are calcified. Most primary tooth germs reach the bell stage and degenerate by embryonic day 22 (E22). Light microscopy on haematoxylin-eosin-stained sections revealed that intensely labelled granular substances are frequently present in the epithelial portion (enamel organs) of the deciduous tooth germs during the period from E18 to E20. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling method, computer-assisted three-dimensional reconstructions, and electron microscopy confirmed that these variable-sized granular substances are similar to apoptotic cells or bodies. Apoptotic structures were mainly found in the primary tooth germ located on the buccal surface of the secondary (successional) tooth germ. These results lead to the conclusion that apoptosis is closely associated with the involution and disappearance of the deciduous tooth germ in S. murinus. A primary tooth germ was observed on the buccal side of all the corresponding successional tooth germs, although the buccal surface of the secondary tooth germ of third upper molar teeth developed only to epithelial thickening without mesenchymal condensation. The findings, therefore, suggest that apoptosis is responsible for disappearance of the primary dental primordia during tooth development in S. murinus.     

氯离子通道ClC-5在大鼠牙胚发育过程中的表达

目的研究氯离子通道ClC- 5在大鼠牙胚发育过程中的表达情况,探讨其在牙齿发育过程中的作用。方法从不同发育阶段的大鼠牙胚中提取总RNA和蛋白,应用RT- PCR和Western blot检测ClC- 5在大鼠牙胚发育过程中mRNA和蛋白的表达情况。结果ClC- 5在牙胚发育的过程中存在时空特异性表达,其mRNA和蛋白都主要在牙胚发育的钟状晚期表达。结论ClC- 5在大鼠牙胚发育过程中的这种时空特异性表达,提示其可能在牙齿发育的过程中发挥一定的作用。     

氯离子通道CIC-5在大鼠牙胚发育过程中的表达

目的研究氯离子通道ClC-5在大鼠牙胚发育过程中的表达情况，探讨其在牙齿发育过程中的作用。方法从不同发育阶段的大鼠牙胚中提取总RNA和蛋白，应用RT—PCR和Westem blot检测ClC-5在大鼠牙胚发育过程中mRNA和蛋白的表达情况。结果ClC-5在牙胚发育的过程中存在时空特异性表达，其mRNA和蛋白都主要在牙胚发育的钟状晚期表达。结论ClC-5在大鼠牙胚发育过程中的这种时空特异性表达，提示其可能在牙齿发育的过程中发挥一定的作用。     

The influence of parathyroid hormone-related protein (PTHrP) on tooth-germ development and osteoclastogenesis in alveolar bone of PTHrP-knock out and wild-type mice in vitro

In a previous study, it was shown that tooth germs of neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are penetrated or compressed by the surrounding alveolar bone, suggesting an important role for PTHrP in the formation and activation of osteoclasts around growing tooth germs. In order to elucidate the role of PTHrP during the development of the tooth germ and related structures, mandibular explants containing cap stage tooth germs of embryonic day 14, homozygous mice were here cultured with or without surrounding alveolar bone. There was no difference in the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells around the first molars of homozygous and wild-type mice. After 10 days of culture, osteoclastic cells were rarely present in explants from homozygous mice and penetration of alveolar bone into the dental papilla was observed. The decline in osteoclast number was partly restored by the addition of PTHrP to the culture. Tooth germs of both wild-type and homozygous mice cultured without alveolar bone developed well, with no apparent structural abnormality; dentine formation was evident after 10 days. These data suggest that PTHrP is not required for the development of the tooth germ proper but is indispensable in promoting the osteoclast formation required to accommodate that development.     

壳聚糖包裹 siRNA 在小鼠牙胚发育研究中的应用

目的：探索局部注射siRNA对小鼠牙胚生长发育产生影响的实验方法。方法：选择健康新生昆明小鼠作为实验对象,将阴性对照siRNA进行壳聚糖（chitosan,CS）包裹,分别将其注入小鼠的两个不同部位：①．腹腔注射组,将Cy5．5标记的CS包裹的siRNA纳米粒注射于新生昆明小鼠腹腔,于不同时问点在动物活体成像仪下观察荧光分布情况。②．颌骨局部注射组,将Cy5．5标记的siRNA/CS纳米粒局部直接注射于新生小鼠左上颌第一磨牙牙胚,通过动物活体成像结合冰冻切片观察荧光分布情况,验证局部注射方法的可行性。结果：在腹腔注射组,随着时间延长,荧光开始在腹腔内扩散,随后在腹腔内淬灭,荧光始终局限于腹腔,腹腔外没有观察到荧光;颌骨局部注射组通过活体动物成像显示注射的牙胚处有荧光,进一步的冰冻切片验证在牙胚处有荧光,且牙胚结构无明显损伤。结论：本实验将CS包裹siRNA纳米粒技术应用于牙胚发育的相关研究,首次成功构建了新生昆明小鼠牙胚局部注射siRNA的方法,为研究牙胚发育相关基因的作用开辟了新的途径。     

维甲酸对小鼠牙胚冠部形态发生的影响

目的探讨维甲酸对早期牙胚形态发生的影响。方法利用体外鼠磨牙牙胚器官培养模型，观察不同剂量维甲酸对１４天胎龄的小鼠下颌第一磨牙牙胚形态发育的影响。结果培养６天后，在不含维甲酸的培养基中牙胚形成明显的牙乳头尖；当培养基中维甲酸的浓度为１×１０－７ｍｏｌ／Ｌ时，牙胚冠部仍形成明显的牙乳头尖；当培养基中维甲酸的浓度为１×１０－６ｍｏｌ／Ｌ时，牙胚冠部无牙乳头尖形成。结论牙胚冠部形态发生与维甲酸剂量有密切关系，过量的维甲酸可抑制牙胚冠部牙乳头尖的形成。     

Cell proliferation in teeth reconstructed from dispersed cells of embryonic tooth germs in a three-dimensional scaffold

Tissue engineering can now reproduce tooth from postnatal tooth cells. However, crown formation is not accurately reconstituted, even when the complex structure of the enamel dentin is reproduced. Here, we showed that a tissue-engineered (TE) tooth, exhibiting morphogenesis according to regular crown-cusp pattern formation, was produced by embryonic tooth germ cells in a three-dimensional scaffold. Heterogeneous cells dissociated from embryonic day 14 (E14) mice tooth germs were seeded on a scaffold and implanted under a kidney capsule in adult mice. The developmental process of the implants was examined for up to 14 d. At 5 d, the cells had formed initial tooth germ, followed by enamel-covered dentin tissue formed symmetrically. To study the developmental process, we examined the growth pattern using 5-bromo-2'-deoxyuridine (BrdU)-labeling analysis. The initial cell-proliferation patterns of the TE teeth were similar to that at the cap and early bell stages in natural teeth. This was particularly true in the cervical loop, which showed a similar distribution pattern of BrdU-positive cells in TE- and natural teeth. These results suggested that even when embryonic tooth germs are dissociated, the single cells can reconstitute tooth, and that enamel organ morphogenesis proceeds as in natural teeth.     

种植牙胚的牙冠发育

目的：观察种植牙胚的牙冠发育情况。方法：选择出生后（PN）8天的SD大鼠,完整剥离第一磨牙牙胚,种植到母鼠肾被膜下,4周后取材观察。结果：种植牙胚的牙冠继续发育,冠周有新生牙槽骨,部分釉质外侧生成骨样结构。结论：牙胚种植到肾被膜下后牙冠可正常发育。