【作者】 李谨；

【导师】 詹瑧；

【作者基本信息】 南京中医药大学 ， 中西医结合基础， 2010， 博士

【摘要】 研究目的：1.进一步明确将斑马鱼作为牙齿发生发育分子生物学研究模型的可行性和先进性。2.研究牙齿发育的重要信号因子（Pax9基因、Pitx2基因）在斑马鱼牙齿萌出与替换过程中的表达部位和时间,以探讨Pax9基因、Pitx2基因在牙发生发育过程中的作用机制及作用途径。3.研究淫羊藿苷对牙齿生长发育的影响。这将加快实现牙齿再生目标,并将为选取中药进行牙齿再生的体内实验提供一定的理论依据。研究方法：1.斑马鱼的饲养及其受精卵的获得：将饲养野生型斑马鱼的水温控制在28.5℃,设定其昼夜规律分别为14小时和10小时。将通过自然交配获得的受精卵饲养于28.5℃水中。斑马鱼的发育阶段按下述方法来划分：孵化期（0—6天龄）；幼虫期（1—4周龄,即7—49天龄）；幼体期（1—3月龄,即30—89天龄）；成熟期（3月龄以上）。2.成年斑马鱼牙齿形态和组织学观察：在体视显微镜下,解剖成年斑马鱼鳃弓,观察其牙齿,并做石蜡切片和HE染色观察。取单个牙齿进行组织学标本处理,扫描电镜下观察其显微结构。3.针对Pax9基因原位杂交探针的制备：根据斑马鱼Pax9基因mRNA序列（GenBank: NM₁31298）设计和合成针对Pax9的引物（F:5’-gcaagcttatggaaatctgcagaggcct; R: 5’-gctggatcccgctcgtcctcctggaa gtagaa）。抽提斑马鱼基因组总RNA,反转录成cDNA,并以此为模板,扩增Pax9基因序列。1%琼脂糖凝胶电泳检测扩增产物。将纯化的PCR扩增产物连接到pGEMT （Promage, Cat.#A3600）载体中,然后转化到DH5α感受态菌株中克隆扩增质粒。将大量扩增抽提得到的连接载体质粒经内切酶消化,在含有地高辛标记的UTP的体外转录系统中,以T7聚合酶催化合成地高辛标记的RNA探针,用于整胚原位杂交。4.Pax9基因在受精72h以内斑马鱼胚胎中的分布和表达：参考Westerfield M. The Zebrafish Book. Eugene:University of Oregon Press,1995.中的整胚原位杂交和胚胎切片方法,取不同发育阶段的胚胎（以4h为观察间隔）,用4%多聚甲醛溶液固定过夜（至少固定12h）,保存于甲醇溶液中,置-20℃备用。用1×PBST溶液洗去多余的甲醇溶液,将胚胎置于65℃水浴箱进行预杂交3h,然后加入所合成的反义RNA探针,65℃水浴杂交过夜。用0.2×SSC溶液洗去多余的探针,加入anti-Dig-AP与反义RNA探针结合过夜。将未结合的抗体用1×PBST溶液洗去,再加入（BCIP/NBT/NTMT）显色30min,迅速用1×PBST溶液洗去多余的显色液,将此胚胎直接在显微镜下观察并记录结果。5.通过制备针对Pitx2基因的探针（同研究方法3）,利用整胚原位杂交技术（同研究方法4）观察Pitx2基因在斑马鱼牙齿早期发育中的表达情况。6.研究淫羊藿苷对牙齿生长发育的影响：在饲养野生型斑马鱼的水中加入淫羊藿苷,观察45天后斑马鱼牙齿生长发育的变化。在扫描电镜下观察其牙齿的显微结构,并对Ca、P等微量元素做能谱分析。7.研究淫羊藿苷对人牙龈成纤维细胞的中药干预作用：对体外培养的人牙龈成纤维细胞（human gingival fibroblasts,HGF）,用MTT法检测不同浓度的淫羊藿苷（0.001、0.01、0.1μg/mL）、不同时间（24、48、72、96h）作用下HGF的增殖水平。研究结果：1、扫描电镜下,斑马鱼牙齿硬组织也具有类似人类牙釉质和牙本质样的结构。人类牙齿的釉质表面有平行排列的与牙长轴垂直的浅凹线纹和不规则的圆形小凹。斑马鱼牙齿表面未见浅凹线纹,但是有一些与牙长轴垂直的颜色较深的条带,在外形变化大的牙尖处尤为明显,这可能同人类牙釉质表面的线纹一样和釉质沉积有关。人类牙齿的牙本质具有牙本质小管,为多孔性组织结构,牙本质构成髓腔,髓腔内充满牙髓等软组织。斑马鱼的釉质内侧组织也呈多孔性疏松组织结构,具有很多贯穿其内的孔隙,与人类牙齿的牙本质结构相似。斑马鱼牙齿的牙本质也围成一髓腔,其内也可见软组织。HE染色进一步观察到红染的牙本质内部为一髓腔,髓腔内具有丰富的牙髓细胞。2、得到了Pax9基因在0到7天各时期斑马鱼胚胎中的表达资料,其中Pax9基因在受精16h的斑马鱼胚胎中开始有特异性信号出现,还可以明显地看到鳃弓（鳃弓,包括牙齿是由神经嵴细胞迁徙分化而来）上的特异性信号从24h-72h的迁徙和发育的过程。3.得到了Pitx2基因在0到7天各时期斑马鱼胚胎中的表达资料,明显地看到鳃弓上的特异性信号从16h-72h的迁徙和发育的过程。4.与对照组相比,实验组（加淫羊藿苷组）中斑马鱼牙齿冠1/3和中1/3的Ca含量显著升高,P含量显著降低,Ca/P比值显著升高。淫羊藿苷对斑马鱼牙齿生长发育中的钙化沉积有明显的促进作用。5.淫羊藿苷（0.001～0.1μg/mL）对人牙龈成纤维细胞（human gingival fibroblasts, HGF）的增殖具有促进作用（P<0.01）,而且呈时间依赖性,促进HGF增殖的最佳浓度为001μg/mL。结论：1.进一步明确了斑马鱼作为牙发生发育分子生物学研究模型的可行性和先进性。从组织结构和超微结构上分析,斑马鱼牙齿和人类牙齿具有相似性,能够作为研究牙齿发生发育的新型模式动物。2.掌握了Pax9基因、Pitx2基因在斑马鱼牙齿发育和替换过程中的的早期表达情况。为揭示Pax9基因、Pitx2基因功能及信号通路等后续研究奠定了基础。3.淫羊藿苷对牙齿生长发育中的钙化沉积有明显的促进作用。淫羊藿苷对人牙龈成纤维细胞有中药干预作用,可以促进牙龈再生。这将为选取中药进行促牙周组织再生的体内实验提供一定的依据。利用中西医结合共同研究牙齿生长发育的机制,加快实现牙齿再生更多还原

【Abstract】 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.更多还原