摘要
牙胚发育早期是牙齿定位与定型的重要阶段。大量研究表明,牙胚发育的起动期决定牙胚形成的位置;帽状期决定牙胚发育的类型,多种生物因子在上皮-间充质间发挥着重要的调控作用。蛋白质组学是一门主要研究蛋白质组的组成成份及生物学功能的新兴学科,从而可以在蛋白水平综合分析影响牙齿发育的调控机制。因此,本实验通过激光捕获显微分离技术分别获取小鼠牙胚发育早期(起动期E11.5天和帽状期E15.5天)的下颌磨牙间充质细胞,采用双向电泳、多级质谱和数据库查询的蛋白质组研究方案,分析小鼠磨牙牙胚发育早期间充质细胞所含蛋白质在数量、种类上的变化,进一步探讨影响牙齿发育的调控因素,以期寻找和发现新的关键因子,从而为牙胚发育研究提供一种新的思路。
目的:1.熟练掌握小鼠牙胚发育起始期、蕾状期、帽状期、钟状期冰冻切片的制备方法;2.熟练掌握激光捕获显微切割技术,并捕获足够量的细胞样本(E11.5天及E15.5天下颌磨牙间充质细胞);3.获得两组细胞的蛋白质双向凝胶电泳图谱,鉴定差异蛋白点,探讨两个时期影响牙胚发育的分子机制。
方法:1.获得准确发育天数的C57胎鼠,制备小鼠下颌磨牙牙胚发育各期的冰冻切片;2.使用PixCell IIe激光捕获显微切割系统对冰冻切片上发育各期的间充质细胞进行分离,收集细胞样本至需要量;3.采用第一向等电聚焦电泳、第二向十二烷基硫酸钠聚丙烯酰胺凝胶电泳、硝酸银染色获得两组细胞样本的双向电泳图,图像分析软件比较差异蛋白点4.利用高效液相色谱-串联质谱仪获得差异蛋白点的一级、二级质谱图,以及蛋白氨基酸序列,通过蛋白质组数据库检索并结合差异蛋白质点的分子信息进行综合分析,鉴定差异蛋白。
结果:1.获得E11.5、12.5、13.5、14.5、15.5、16.5、17.5天小鼠下颌磨牙牙胚冰冻切片;2.经激光捕获得到需要量的两组(E11.5牙胚起动期、E15.5牙胚帽状期)下颌磨牙间充质细胞;3.两组细胞双向电泳图获得20个差异蛋白点,其中15个点得到鉴定。质谱结果显示:E11.5组细胞新增蛋白为膜联蛋白A2、肌动蛋白β、丙酮酸激酶M;E11.5组细胞上调蛋白为ATP合成酶d亚单位、ATP合成酶α亚单位、转酮酶、β烯醇酶;E15.5组细胞新增蛋白为真核翻译延长因子2、乳酸脱氢酶B、M-Septin;E15.5组细胞上调蛋白为真核翻译延长因子1γ、核内核糖核蛋白H2、核内核糖核蛋白X、乳酸脱氢酶A、丝氨酸/苏氨酸蛋白磷酸酯酶1γ。
结论:1.通过阴栓观察及胎鼠身长测量相结合的方法,提高了胎龄判断的准确性和小鼠的利用率;2.经过对发育各阶段胎鼠头部进行连续冰冻切片,明确下颌磨牙牙胚的具体位置,大大提高了获得牙胚冰冻切片的效率;3.通过激光捕获显微切割技术成功获得目的细胞;4.通过差异蛋白点的鉴定及分析,获得的蛋白主要为细胞糖代谢及能量代谢相关蛋白酶、参与蛋白翻译合成、以及参与某些信号传导途径的信号分子等,这些蛋白分别在小鼠下颌磨牙牙胚发育起动期和帽状期间充质细胞中存在特异性表达或表达上调,为进一步研究牙胚早期发育的调控机制提供了新的线索。
Early tooth development is an important stage determining tooth region and type. Many researches indicated that initiation stage of tooth development determined the tooth region and cap stage determined the tooth type. Signals regulating between epithelial and mesenchymal tissues have made an very important function during the two stages. Proteomics is a new science that investigates the protein composition and function of proteome.The regulating mechanisms of tooth development can be studied on the level of proteome. Therefore in the present study, we captured the mesenchymal cells of mouse lower molar germ at E11.5d (initiantion stage) and E15.5d (cap stage) by laser capture microdissection (LCM). The differentially expressed proteins of the mesenchymal cells at the two stages above-mentioned were analyzed on quantity and quality by two-dimensional gel electrophoresis, mass spectrometry, data base querying technology. The aim of our study was to further investigate the regulating factors, even to find some new signals expressed in the tooth germ and to provide a new method on the mechanisms research of tooth development.
Objectives: 1. To grasp practisedly the preparative method of the frozen sections of mouse germs at different stages(initiation, bud, cap, bell stage) during tooth development; 2. To grasp practisedly the techniqu of LCM and obtain the sufficient amount of cell samples; 3. To establish the atlas of protein two-dimensional gel electrophoresis of the mesenchymal cells in the mouse lower molar germs at the stages of E11.5d and E15.5d. To identify the differently expressed proteins and investigate the molecular mechanisms of tooth development during the two stages above-mentioned.
Methods: 1. C57 pregnant mice that had accurate fetal ages were obtained and the frozen sections of mouse germs at different stages during tooth development were prepared; 2. The mesenchymal cells on the frozen sections by using PixCell IIe were obtained and sufficient cells of each sample were collected; 3. The atlas of protein two-dimensional gel electrophoresis (2-DE) of samples were obtained by isoelectric focusing electrophoresis (IEF), sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and AgNO3 staining. The differently expressed proteins were analyzied by image analysis software; 4. First, second class mass-spectrograms and amino acid sequences of proteins were obtained by high performance liquid chromatogram- mass spectrum/ mass spectrum (HPLC-MS/MS). The differently expressed proteins were identifid by protein database querying and protein information analysis.
Results: 1. The frozen sections of mouse lower molar germs at E11.5、12.5、13.5、14.5、15.5、16.5、17.5d during tooth development were prepared; 2. We have obtained sufficent mesenchymal cells of mouse lower molar germs at E11.5d and 15.5d; 3. We have obtained 20 differently expressed proteins on the atlas of 2-DE and 15 prteins were identified. Annexin A2,β-actin, pyruvate kinase M were expressed specificly in the sample of E11.5d; ATP synthase subunit d, ATP synthase alpha subunit, transketolase, beta-enolase were up-regulatedly expressed in the sample of E11.5d; Eukaryotic translation elongation factor 2, lactate dehydrogenase B, M-Septin(also named Septin 4)were expressed specificly in the sample of E15.5d;Eukaryotic translation elongation factor 1gamma, heterogeneous nuclear ribonucleoprotein H2, heterogeneous nuclear ribonucleoprotein X, lactate dehydrogenase A, serine/threonine-protein phosphatase 1gamma were up-regulatedly expressed in the sample of E15.5d.
Conclusions: 1. The fetal age could be judged more accuratly and the use ratio of mice could be increased by the combination of observing pessulum and measuring fetal mouse body length; 2. The regoin of lower molar germ in the head had been knowed by using the method of cutting the frozen head embed of different fetal age mice one by one section. Then the efficiency of obtaining the frozen sections of tooth germs was markedly increased; 3. We have obtained the mesenchymal cells from the frozen sections successfully by using LCM;4. Using proteomics methods we have obtained some defferently expressed proteins between two samples.By identifying , the proteins mainly comprised some enzymes correlated with glycometabolism and energy metabolism in the cells, some factors related with the protein translation and some molecules correlated with some signal conduction pathways. They were specificly or up-regulatedly expressed in the mesenchymal cells of mouse lower molar germs at initiation stage and cap stage respectively, which provided a new clue for the research on the regulating mechanisms of tooth development during early stages.
引文
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