Signal Regulatory Mechanisms And Effect Of Icariin During Tooth Generation

Objective:1. Further to confirm the feasibility and advantages of zebrafish being used as a model animal to study the molecular biological mechanisms during tooth generation and development.2. To identify the expression sites and time-points of important signal factors of genes Pax9 and Pitx2, to discuss their mechanisms and pathways of action during tooth generation and development.3. To study the effects of Icariin on tooth development in order to provide some evidence for in vivo experiments on tooth regeneration with Chinese traditional medicine, and to expedite the achievement of tooth regeneration.Methods:1. Zebrafish breed and fertilized eggs obtained from it:The rearing water temperature for the wild-type zebrafish was set at 28.5℃, and the length of day and night were set at 14 h and 10 h, respectively. Then, the eggs fertilized through natural mating were bred in 28.5℃water. The developmental stages of zebrafish are divided as:embryo stage (0-6 days old), larval stage (1-4 weeks old), pupal stage (1-3 months old) and imago stage (more than 3 months old).2. Mature zebrafish tooth observation with morphological and histological methods:Under stereomicroscope, gill-arch of mature zebrafish was dissectedand the teeth were observed in vivo, paraffin sections were made and HE stain was observed. Then a single specimen of teeth was taken for histological processing and observed its microstructure with scanning electron microscope (SEM).3. DNA probes of gene Pax9 preparation for the in situ hybridization:Based on the mRNA sequence of Pax9 in zebrafish (GenBank:NM₁31298), primers for Pax9 (F:5'-gcaagcttatggaaatctgcagaggcct; R:5'-gctggatcccgctcgtcctcctggaa gtagaa) Were designed and synthesized. The total RNA of zebrafish genome was extracted, and reverse-transcripted them into cDNA as the template for amplifying sequence of Pax9. The products were tested by electrophoresis with 1% agarose gel. Purified PCR products with vector pGEMT (Promage, Cat.# A3600) were combined and, then transformed into feeling-state strain DH5a to expanse the plasmids. The connecting-vector plasmids were digested, which were abundantly amplified and extracted, using incision enzyme. Under in vitro transcription system containing digoxin-labeled UTP, the digoxigenin-labeled RNA probes, synthesized under catalysis of T7 polymerase, were used in whole mount in situ hybridization.4. Pax9 distribution and expression in zebrafish embryos fertilized within 72h:According to the methods of whole embryo in situ hybridization and embryo biopsy in The Zebrafish Book (Westerfield M. The Zebrafish Book. Eugene:University of Oregon Press, 1995.), took embryos at different developmental stages (set the observing interval at 4h), fixed the samples with 4% paraformaldehyde solution overnight (at least for 12h), and conserved the samples in methanol at -20℃. Scoured off the excess methanol with 1×PBST solution, put the embryos into water-bath at 65℃to pre-hybridize for 3h. Then added antisense RNA probes, and hybridized in 65℃water bath overnight. Washed away excess probes with 0.2×SSC solution, then added anti-Dig-AP to combine with the antisense RNA probes overnight. We flushed away the unconjugated antibodies with 1×PBST solution, colorated for 30min with colorating liquid (BCIP/NBT/NTMT), washed out the excess colorating liquid with 1×PBST immediately and directly observed the embryos under microscope.5. DNA probes preparation for Pitx2, and identified its expressions in zebrafish teeth in early developmental stages by whole embryo in situ hybridization. (The methods were similar as for gene Pax9.)6. Study the effect of Icariin during tooth generation:Icariin was added into the rearing water for wild-type zebrefish to detect the changes of tooth generation in 45 days. Micro structure of the teeth, and operated spectrum analysis on Ca, P and other trace elements were observed under SEM.7. Study the effect of Icariin on human gingival fibroblasts (HGF):Using MTT assay to detect the proliferation level of human gingival fibroblasts, which were cultured in vitro, with different Icariin densities (0.001,0.01,0.1μg/mL) at 24,48,72, and 96 h, respectively.Results:1. Under SEM, zebrafish teeth have similar enamel and dentin structure as human teeth. The enamel surface of human teeth have parallel dimples, with are vertical to the tooth prolate axis, and irregular round concaves. Although no dimples were found on the surface of zebrafish teeth, several dark straps were detected, which were vertical to the prolate axis, especially at the apex cuspids where the shape changes notably. The straps might also be related to enamel deposition as the strikes on the surface of human enamel. Dentin of human teeth has dentinal tubules, so the structure is porous. Tooth cavity, surrounded by dentin, is filled with soft tissues as pulp. The inner structure of zebrafish enamel is also porous as human enamel, with lots of penetrating ventages. Dentin of zebrafish teeth also forms a cavity, containing soft tissue. Tissues in the cavity, by HE staining, contain plenty of pulp cells were found.2. The expression of Pax9 at different stages in zebrafish embryos in days 0-7 were obtained. Specific signals of Pax9 appeared in the embryos at 16 h. Also, the specific signals in the procedure of migration and differentiation in gill-arch (Gill-arch, including tooth, migrates and differentiate from neural crest cells.) during 24-72 h were found.3. The expressions of Pitx2 in zebrafish embryos during days 0-7 was obtained. It's obvious that specific signals in the procedure of migration and differentiation in gill-arch at 16-72h.4. Comparing with the control group, teeth in the experimental group contained notably larger amount of Ca and less P in the crown third and the middle third. As a result, the ratio of Ca/P obviously high. We believed Icariin could strongly promote calcium deposition during tooth generation.5. Icariin at 0.001～0.1μg/mL could promote the proliferation of HGF (P<0.01), with a time-dependent property. The optimum density for promoting HGF proliferation is 0.01μg/mL.Conclusion:1. It is further confirmed the feasibility and advantages of zebrafish being used as a model animal to study molecular biological mechanisms during tooth generation and development. In the aspects of organizational structure and ultramicrostructure, zebrafish teeth have much similarity with human teeth, and zebrafish can be used as novel model animal to study tooth generation.2. It is clarified the expressing situation of Pax9 and Pitx2 in the early stages of zebrafish tooth generation and replacement, and have laid foundation for subsequent researches about functions and signal pathways of gene Pax9 and Pitx23. Icariin could greatly promote the procedure of calcium deposition during tooth generation and could improve gingival regeneration's effect on human gingival fibroblasts. This study would provide some support for in vivo experiments on periodontal tissue regeneration with Chinese traditional medicine. By combining Chinese traditional medicine and Western Medicine to study the tooth regeneration mechanisms at the molecular biology level, could expedite the achievement of tooth regeneration.