摘要
牙齿的发育研究可以为牙齿再生及牙齿先天发育异常提供理论依据,同时牙齿发育也是发育生物学中器官发育形成的一个典型模型。目前牙齿早期发育研究较多,但后期的发育,特别是牙根发育相关基因研究较少。牙齿发育是口腔上皮组织与间充质组织相互作用的结果,牙根的发育与上皮根鞘有着密切关系,当牙冠发育即将完成之际上皮根鞘的形成揭开了牙根发育的序幕。本研究目的是利用大鼠下颌磨牙和切牙具有完全不同的牙根发育模式作为动物模型,对牙根发育相关的差异表达基因进行筛选并对部分差异基因的功能进行探讨,为牙根发育及牙根再生研究提供理论依据。
采用的方法及主要结果:本课题采用组织学、免疫组织化学、改良消减杂交技术、Northern blot、Western blot、RT-PCR、Real-time PCR、细胞培养、基因转染和RNA干扰等方法,进行了以下五部分实验研究:
第一部分 通过组织学观察大鼠下颌第一磨牙和下颌切牙的发育时序,结果发现胚胎18日(E18)大鼠磨牙和切牙发育开始出现差异,这种差异的表现是磨牙形成了双层细胞的上皮根鞘,开始启动牙根的发育,而切牙舌侧虽然也形成类似根鞘的结构,唇侧却仍然维持多层结构的颈环干细胞龛,始终未能启动牙根发育,仍旧保持牙冠的发育形式。
第二部分 实验1通过改良消减杂交技术,首次建立了大鼠牙根发育启动相关差异表达基因文库,文库总浓度约为1.5×10~4,经酶切鉴定含有500bp以上插入片段的克隆约为84%,表明所建立的基因文库质量较高,
Objectives: Tooth development is a fine research model to study the interactions between epithelium and underlying mesenchymal. From studying the tooth development mechanism, we will not only find the origin of clinical congenital tooth abnormity but also make it possible to achieve the tooth regeneration, which now is still a dream of human. Compared with many studies of early tooth development, little was known about the root development. The root development begins at the forming of Hertwig's epithelial root sheath which was double layers of cells, and the rat molars and incisors have different root patterning mechanisms: the former formed a multirooted pattern while the latter formed a single-rooted analogue. In this study, we use the natural tooth root development animal model—the rat incisor and molar root patterning to screening the root patterning related genes and study some of the genes' functions in development. Methods and the main results: By using molecular biology and cell biology technologies, such as substractive hybridization, northern blot, Western blot ,RT-PCR, Real-time PCR, cell culture, gene transfection and RNAi, this study obtained the following results:
In the section 1, we first observed the Hertwig's epithelial root sheath's development timing of molar and incisor, and we founded that in E18 the molar and the incisor began differentially development: the molar formed double-layer cells of root sheaths, but the incisor labial epithelium grew longer in length than the lingual epithelium and formed the cervical loop, which was a region thought to contain a population of self-renewing epithelial stem cells. Though the lingual epithelium formed double layers of root sheath, the incisor germs continue to grow as crown pattern without forming a root.In the section 2 study 1, based on the section 1, the rat (E18) lower molar and incisor tissue were removed and mRNA was abtained by magnetic beads coupled with oligodT, and the mRNA were synthesized and amplified by SMART PCR cDNA Synthesis Kit. Then, using MSH method and T/A cloning techniques, cDNA fragments of differentially expressed genes in rat molar were inserted into competence DH5a bacteria. Positive clone were selected randomly and evaluate by enzyme digestion. cDNA subtractive library of rat molar E18 was set up successfully with high subtractive efficiency. 84% positive clone were identified by enzyme digestion and most of the insert were longer than 500 bp. the library's total content was about 1.5×10~4. In the study 2, by using reverse dot blot and DNA sequencing, we screened the positive clones and found some development related genes which were differentially expressed in the molar and incisors and were strongly related the tooth initiation and root patterning, such as Se11L, NfiC, Edar, GATA6, and some new genes or unknown genes. We successfully constructed a subtraction cDNA library of rat molar root developmental patterning, which laid a foundation for studying specific genes for tooth root developmental patterning.
In the section 3 study 1, we named a new EST "Molar Root Patterning gene 1" (Mrp1) and submitted to the GeneBank with a accession number AY598353.We Designed a Mrp1-specific DNA probe and by using Northern blot and RT-PCR, we found that the expression of Mrpl were not only in the molar but also in the developing pancreas, liver,lung and kidney tissues, and the Mrpl mRNA full length was about 3Kb. By using bioinformatics analysis, we found the Mrp1 was located in the 18q12.3 chromosome of the rats and the Mrp1 amino acids sequence had about 37% homology with a known protein Uroplakin IIIb (p35) which was an urothelial differentiation membrane molecular marker. And by other biological software we found Mrpl had a trans-membrane structure and 5 PKC phosphorylation sites, 4 CKII phosphorylation sites.In the study 2, we successfully constructed a recombinant prokaryotic expression vector PGEX-4T-Mrpl which expressed the C-terminal polypeptide of Mrp1 in the E.coli D H 5a bacteria.In the section 4, we construct and analysis multiple alternative spliceosome of NfiC gene of E18 rat molar tissues, because previous studies had shown that NfiC gene was specific to the tooth root development and play a key role in the root formation, and NfiC gene was differentially expressed in molar and incisor germs. Two pairs of primers were designed, which were specific to the NfiC gene and by RT-PCR method, we amplified the NfiC gene in the rat molar tissue. The fragments were were inserted into competence DH5a bacteria. Positive clone were selected randomly and evaluated by enzyme digestion and sequencing. As the results that we found that there were at least three deffierent alternative spliceosome of NfiC gene in the rat molar which were named with rNFIC-1、 rNFIC-2 and rNFIC-3.As a conclusion from section 4, the NfiC gene expressed in the rat molar, and there was a multiple
alternative splicing way for the NfiC gene to play their functions which was unknown in the tooth development.In the section 5 study 1, in order to study the root patterning related gene Sel1L in the tooth development, first we studied the Sel1L's expression pattern in the tooth development and in vitro cultured Tooth Germ Cells(TGCs) using a monocloned antibody of Sel1L(a generous gift from Dr.Sylvie Menard and Dr.Ida Biunno).the immunohistochemistry results proved the SellL had a dynamic expression pattern in the tooth development: in the early stage, it was only a weak expression in the dental epithelial and neighbouring mesenchymal., but in the E18(root pattern period), it was mainly located in the differentiated cells, such as ameoblasts and odontoblasts, and in the incisor, only the cervical loop marginal cells were positive and the inner epithelia stem cells were not positive which inferred the SellL might related with cells' differentiation. In the study 2, we constructed a RNAi vector which specifically knock down the SellL gene's expression and transfected into TGCs. After transfection, we detected the SellL expression level by RT-PCR and western blot, the results showed RNAi vector had a nice blocking effect of SellL gene. In the study3, we studied the phenotype and genotype of TGCs after RNAi vector transfection. The results showed that compared with the control group: (1)the transfected cell had a enhanced proliferation which demonstrated SellL gene might inhibit the cells' growing.(2)the transfected group cells had less apoptosis.(3)by using RT-PCR and real-time PCR, we found the Hesl gene, which were the main targets of Notch signaling, were up-regulated after RNAi.(4)Western blot results showed Smad4, the TGFβ signaling molecular, were down-regulated.(5)Western blot results also showed DSPP, the chief differentiation marker of odontoblasts, signaling molecular,
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