CCN3通过Notch与BMP信号通路调控牙髓干细胞在牙再生中增殖与分化的研究

发布时间：2018-04-23 23:17

  本文选题：牙再生 + 肾母细胞瘤过表达因子(NOV/CCN3)　； 参考：《第三军医大学》2014年博士论文

【摘要】：研究背景： 在牙发育过程中及牙成熟后，牙本质生成由终末成体成牙本质细胞分泌所形成。第一牙本质在牙冠和牙根发育成形过程中由成牙本质细胞分泌而成。第二牙本质生成于牙根发育成形，并且其分泌速度明显减慢。当牙发育完成，牙本质-牙髓复合体维持着牙内平稳状态以及自我保护机制，在牙损伤后形成保护牙髓的修复性牙本质层，，即第三牙本质。成熟后的牙髓中含有干细胞，负责修复龋和机械磨损刺激给牙体牙髓造成的损伤及缺损。牙髓干细胞(Dental pulp stem cells, DPSCs)，具有单克隆能力，高效自我更新活性及多向分化能力，已被公认为在牙组织修复过程中起着至关重要的作用。在牙受损后，DPSCs发生募集，移行，增殖并且向成牙本质细胞方向分化，最终在牙损伤处合成分泌出第三牙本质。 修复性牙本质的形成由众多信号通路精密控制形成。近几年，牙本质形成的细胞、分子机制已成为牙再生治疗方法和临床研究探索的热点。牙在发育形成过程中，众多信号通路参与了上皮-间充质相互作用。Notch受体及配体在牙发育成形过程中表达于上皮和间充质组织。Notch信号通路已被证实在决定牙内皮细胞的命运及成牙本质分化方向方面起着至关重要的作用。Notch受体正常成体牙髓组织中表达较弱，而在牙体受损后其表达显著增强。在成体牙髓中，Notch1表达于干细胞部位，Notch2表达于成牙本质前体细胞及成牙本质细胞。骨形成蛋白(bone morphogenetic protein，BMPs)已被证实为诱导牙内皮-间充质相互作用中关键的信号通路。BMP2转录存在于牙乳头，并且随着成牙本质细胞的分化过程急剧增加。炎症作为一种牙损伤后的反应，与牙再生的过程密切相关。炎症产物在牙损伤部位与BMPs相互作用以促进牙损伤的修复与治愈。但是，对Notch与BMPs信号通路在牙修复再生过程中促进DPSCs增殖与分化的关键启动因子研究尚不清楚。 NOV/CCN3(Nephroblastoma overexpressed)最先被认为是人造血干细胞前体细胞启动因子。CCN3被敲除使原始前体细胞丧失功能。CCN3还被发现与组织愈合过程*本课题受国家自然科学基金项目（81170934；81300873）资助。密切相关。CCN3高度表达于皮肤伤口的肉芽组织，并且诱导纤维母细胞的一系列反应。因此CCN3被设想为有可能活跃地参与DPSCs功能的调节，并且在牙组织再生中起到重要的调控作用。 方法： 为研究第三牙本质在损伤条件下的反应情况，利用柱型金刚石钻头在无菌条件下制备成暴露的大鼠切牙缺损模型，并标记出牙髓中特异性表达CCN3的细胞群。接着利用慢病毒介导的CCN3过表达载体感染人DPSCs.通过细胞周期分析，CCK-8检测和PCNA表达检测研究了过表达CCN3对DPSCs增殖的影响。利用碱性磷酸酶检测，茜素红染色和细胞钙浓度定量分析检测过表达CCN3对人DPSCs成牙本质分化的影响。通过qRT-PCR和Western-blot检测Notch信号通路以及Runx2的表达。BMP2是诱导DPSCs成牙本质分化的重要因子，通过qRT-PCR和Western-blot检测了其表达。另外，将CCN3过表达DPSCs与对照组DPSCs以1：4混合在成牙本质分化诱导条件下共培养。通过碱性磷酸酶活性检测，茜素红染色和细胞钙浓度定量分析检测了共培养组细胞的成牙本质分化能力。CCN3过表达DPSCs与对照组DPSCs混合植入多孔PLGA支架进行体外成牙本质分化诱导2周后，将此细胞支架复合物移植到裸鼠皮下，进行von kossa染色检测其矿化能力。最后，BMP2以3.5nM浓度做用DPSCs，通过qRT-PCR及Western-blot方法检测DPSCs中CCN3表达情况。 结果： 在牙修复再生过程中，CCN3呈现出短暂的高表达。过表达CCN3通过上调Notch信号通路促进人DPSCs增殖，并且抑制DPSCs成牙本质分化。过表达CCN3还上调DPSCs中BMP2表达以及分泌。过表达CCN3DPSCs通过上调BMP2分泌促使对照DPSCs以非自主方式上调成牙本质分化能力，并且裸鼠皮下移植实验证实其矿化能力增强。同时，BMP2对正常DPSCs中CCN3及Notch信号通路起抑制作用。 结论： 1.此研究表明，CCN3特异地表达于牙髓组织内干细胞并且在损伤后牙再生修复开始时期表达明显上调。 2.过表达CCN3通过上调Notch信号通路增强了DPSCs增殖能力并且抑制其成牙本质分化能力。 3. CCN3促进DPSCs中BMP2的表达及分泌，但被上调的Notch信号通路抑制了BMP2对DPSCs的促成牙本质分化作用。 4.过表达CCN3促使DPSCs中BMP2分泌增强，此BMP2以非细胞自主方式促进DPSCs成牙本质分化。 5.体内试验中，由过表达CCN3的DPSCs和正常DPSCs以1：4比例混合共培养系统里，CCN3对促进牙再生起到至关重要的作用。
[Abstract]:Research background:
In the process of tooth development and tooth maturation, dentin formation is formed by the secretion of end-end adult odontoblast cells. The first dentin is secreted by odontoblast cells during the process of forming the crown and root. The second dentin is formed by the formation of the root development and its secretory speed is obviously slowed down. The intramedullary complex maintains a stationary state and a self-protection mechanism, forming a restorative dentin that protects the dental pulp after dental injury. The third dentin, which contains stem cells in the mature pulp, is responsible for repairing caries and mechanical attrition to the dental pulp. Dental pulp stem cells, DPSCs McAb, high efficient self renewal activity and multidirectional differentiation, has been recognized as a vital role in dental repair. After tooth damage, DPSCs is raised, migrated, proliferated and differentiated into dentin cells. Finally, the third dentine is synthesized and secreted at the tooth damage.
The formation of restorative dentin is precisely controlled by a number of signal pathways. In recent years, the molecular mechanism of dentin formation has become a hot spot in dental regeneration treatment and clinical research. In the process of tooth development, many signal pathways have participated in the epithelial mesenchymal interaction.Notch receptor and ligand in tooth development. The.Notch signaling pathway expressed in epithelial and mesenchymal tissue has been proved to play a crucial role in determining the fate of dental endothelial cells and the direction of dentin differentiation. The expression of.Notch receptor in normal adult dental pulp tissue is weaker, and its expression is significantly enhanced after the tooth is damaged. In the adult dental pulp, Notch1 is expressed in the stem. The cell site, Notch2 is expressed in dentin precursor cells and odontoblast cells. Bone morphogenetic protein (BMPs) has been proved to be the key signaling pathway of the induced endothelium - mesenchyme interaction,.BMP2 transcription in dental papilla, and as the process of odontoblast differentiation increases rapidly. The response of a tooth after injury is closely related to the process of tooth regeneration. The inflammatory products interact with BMPs to promote the repair and cure of tooth damage. However, the study of the key promoter of the Notch and BMPs signaling pathway to promote the proliferation and differentiation of DPSCs in the process of tooth repair and regeneration is not clear.
NOV/CCN3 (Nephroblastoma overexpressed) was first considered to be the knockout of the precursor cell promoter.CCN3 of artificial blood stem cells to make the original precursor cell loss of function.CCN3 found and tissue healing * this subject was funded by the National Natural Science Foundation (81170934; 81300873). Closely related.CCN3 is highly expressed in skin wounds. The granulation tissue and a series of responses to the fibroblasts are induced. Therefore, CCN3 is envisaged as potentially active in the regulation of DPSCs function and plays an important role in dental tissue regeneration.
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