| Background: There are only two dentitions in our life. After the primary dentition being replaced by permanent dentition during 12 to 13 years old, not any teeth will come out to replace the permanent dentition. However, our teeth will get wear out with using them. A healthy dentition is the foundation of our digestion system. Nevertheless, with the development of society and diversity of food, series of congenital or extrinsic reasons inactivate the function of tooth. In case we lost our permanent teeth, it means that they are separate from us forever,and we cannot get some kind of regenerative teeth. Oligodontia is not only a threat to our health but also a block may lead to mental disorder. Nowadays, some scholars name the implant denture as the third dentition of human beings. Admittedly, implant denture makes the minimum damage contrast with the traditional ways of dental restoration. Inevitably, however, implant teeth have some disadvantages such as high expectation of health condition, high cost and long period of restoration. With the raising of people’s consciousness of oral health, the real third dentition not the denture has become the difficult point and focus of dental community. So the development of dentition and regeneration of teeth has been grown into a hot-topic subject.Most scientists chose the mice, black small-eared pigs and dogs as model animals in survey about teeth development and regeneration in recent years. Extensive studies brought us to a new world. Now we know something concerning tooth formation, migration and differentiation of cells, interactions between organizations. What’s more, to some extent, we learned the mineralization of hard tissue and the extension of root. Nevertheless, there are some drawbacks of mammals. Because local tissue is buried deep under the skin or connective tissue, it’s quite difficult to observe these tissues. If we want to do some experiment in an early stage of embryos, we must take the body out of its mother’s uterus. Zebrafish is a new kind of oviparity model animal. Lots of merits are found in zebrafish, so we choose zebrafish as our laboratory animals during this experiment.We add some exogenous retinoic acid into egg water to establish an excessive RA model. And we add synthetase inhibitor of raldh2 to egg water in order to create a deficiency of RA. By the same time, heat shock protein is used to promote the catabolic enzyme of RA cyp26a1 so as to build an endogenous absence of RA. After drug treatment, whole mount in situ hybridization is used to check the specific protein of teeth formation. Tooth epithelium specific fluorescence protein is used to observe the fluorescence at the same time.Objective: To get a recombinant plasmid of cyp26a1.To construct a transgenic line that can overexpression RA catabolic enzyme cyp26a1.Check the infection of excessive RA and absence of RA in teeth development.Methods: We cloned the coding sequence of cyp26a1 from cDNA library. Then we connected it into the HSP plasmid. We injected this plasmid into the animal pole of one single cell stage embryos. After screening for about three generations, we will acquire homozygote of transgenic line. Meanwhile, we cloned four specific marker of neural crest, pharyngeal arch and teeth. We wanted get mRNA probe of dlx2 a, dlx2 b, scpp5 and barx1. We utilized concentration gradient RA and DEAB to treat Tg:dlx2b-GFP and AB Go wildtype zebrafish. By the same time, we used heat shock to make cyp26a1 overexpression wildly. In the end, we used probe to check these specific protein expression.Results:We got the recombinant plasmid of cyp26a1 successfully. Then the transgenic line Tg(Hsp-cyp26a1-GFP; cryaa-Venus) zebrafish has been successfully established. We obtained three mRNA probes successfully. Compared with DMSO control group, a low concentration(1×10-7M) of RA could up-regulate the expression of mRNA(barx1, dlx2a) in neural crest. Obvious migration trend was observed toward the pharyngeal arch in which teeth adhered. Transgenic fish had spreading fluorescence tendency in pharyngeal arch. At the same time, a high concentration(in this experiment we mean 4×10-7M) of RA malformed the embryos and killed them after treatment. One third of the embryos of middle concentration(3×10-7M) exhibited delayed development. DEAB resulted in neural crest dysplasia. The expression of barx1 and dlx2 a were suppressed, and the appearance of dlx2 b in tooth was delayed. The results of Tg: Hsp-cyp26a1-GFP; cryaa-Venus heat shock is similar to the DEAB group.Conclusion: RA signal pathway can regulate the progenitors of tooth by controlling the growth of the neural crest and manipulating tooth development. |
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