本文選題：牙再生 + 腎母細胞瘤過表達因子(NOV/CCN3)�。� 參考：《第三軍醫(yī)大學》2014年博士論文

【摘要】：研究背景： 在牙發(fā)育過程中及牙成熟后，牙本質生成由終末成體成牙本質細胞分泌所形成。第一牙本質在牙冠和牙根發(fā)育成形過程中由成牙本質細胞分泌而成。第二牙本質生成于牙根發(fā)育成形，并且其分泌速度明顯減慢。當牙發(fā)育完成，牙本質-牙髓復合體維持著牙內(nèi)平穩(wěn)狀態(tài)以及自我保護機制，在牙損傷后形成保護牙髓的修復性牙本質層，，即第三牙本質。成熟后的牙髓中含有干細胞，負責修復齲和機械磨損刺激給牙體牙髓造成的損傷及缺損。牙髓干細胞(Dental pulp stem cells, DPSCs)，具有單克隆能力，高效自我更新活性及多向分化能力，已被公認為在牙組織修復過程中起著至關重要的作用。在牙受損后，DPSCs發(fā)生募集，移行，增殖并且向成牙本質細胞方向分化，最終在牙損傷處合成分泌出第三牙本質。 修復性牙本質的形成由眾多信號通路精密控制形成。近幾年，牙本質形成的細胞、分子機制已成為牙再生治療方法和臨床研究探索的熱點。牙在發(fā)育形成過程中，眾多信號通路參與了上皮-間充質相互作用。Notch受體及配體在牙發(fā)育成形過程中表達于上皮和間充質組織。Notch信號通路已被證實在決定牙內(nèi)皮細胞的命運及成牙本質分化方向方面起著至關重要的作用。Notch受體正常成體牙髓組織中表達較弱，而在牙體受損后其表達顯著增強。在成體牙髓中，Notch1表達于干細胞部位，Notch2表達于成牙本質前體細胞及成牙本質細胞。骨形成蛋白(bone morphogenetic protein，BMPs)已被證實為誘導牙內(nèi)皮-間充質相互作用中關鍵的信號通路。BMP2轉錄存在于牙乳頭，并且隨著成牙本質細胞的分化過程急劇增加。炎癥作為一種牙損傷后的反應，與牙再生的過程密切相關。炎癥產(chǎn)物在牙損傷部位與BMPs相互作用以促進牙損傷的修復與治愈。但是，對Notch與BMPs信號通路在牙修復再生過程中促進DPSCs增殖與分化的關鍵啟動因子研究尚不清楚。 NOV/CCN3(Nephroblastoma overexpressed)最先被認為是人造血干細胞前體細胞啟動因子。CCN3被敲除使原始前體細胞喪失功能。CCN3還被發(fā)現(xiàn)與組織愈合過程*本課題受國家自然科學基金項目（81170934；81300873）資助。密切相關。CCN3高度表達于皮膚傷口的肉芽組織，并且誘導纖維母細胞的一系列反應。因此CCN3被設想為有可能活躍地參與DPSCs功能的調節(jié)，并且在牙組織再生中起到重要的調控作用。 方法： 為研究第三牙本質在損傷條件下的反應情況，利用柱型金剛石鉆頭在無菌條件下制備成暴露的大鼠切牙缺損模型，并標記出牙髓中特異性表達CCN3的細胞群。接著利用慢病毒介導的CCN3過表達載體感染人DPSCs.通過細胞周期分析，CCK-8檢測和PCNA表達檢測研究了過表達CCN3對DPSCs增殖的影響。利用堿性磷酸酶檢測，茜素紅染色和細胞鈣濃度定量分析檢測過表達CCN3對人DPSCs成牙本質分化的影響。通過qRT-PCR和Western-blot檢測Notch信號通路以及Runx2的表達。BMP2是誘導DPSCs成牙本質分化的重要因子，通過qRT-PCR和Western-blot檢測了其表達。另外，將CCN3過表達DPSCs與對照組DPSCs以1：4混合在成牙本質分化誘導條件下共培養(yǎng)。通過堿性磷酸酶活性檢測，茜素紅染色和細胞鈣濃度定量分析檢測了共培養(yǎng)組細胞的成牙本質分化能力。CCN3過表達DPSCs與對照組DPSCs混合植入多孔PLGA支架進行體外成牙本質分化誘導2周后，將此細胞支架復合物移植到裸鼠皮下，進行von kossa染色檢測其礦化能力。最后，BMP2以3.5nM濃度做用DPSCs，通過qRT-PCR及Western-blot方法檢測DPSCs中CCN3表達情況。 結果： 在牙修復再生過程中，CCN3呈現(xiàn)出短暫的高表達。過表達CCN3通過上調Notch信號通路促進人DPSCs增殖，并且抑制DPSCs成牙本質分化。過表達CCN3還上調DPSCs中BMP2表達以及分泌。過表達CCN3DPSCs通過上調BMP2分泌促使對照DPSCs以非自主方式上調成牙本質分化能力，并且裸鼠皮下移植實驗證實其礦化能力增強。同時，BMP2對正常DPSCs中CCN3及Notch信號通路起抑制作用。 結論： 1.此研究表明，CCN3特異地表達于牙髓組織內(nèi)干細胞并且在損傷后牙再生修復開始時期表達明顯上調。 2.過表達CCN3通過上調Notch信號通路增強了DPSCs增殖能力并且抑制其成牙本質分化能力。 3. CCN3促進DPSCs中BMP2的表達及分泌，但被上調的Notch信號通路抑制了BMP2對DPSCs的促成牙本質分化作用。 4.過表達CCN3促使DPSCs中BMP2分泌增強，此BMP2以非細胞自主方式促進DPSCs成牙本質分化。 5.體內(nèi)試驗中，由過表達CCN3的DPSCs和正常DPSCs以1：4比例混合共培養(yǎng)系統(tǒng)里，CCN3對促進牙再生起到至關重要的作用。
[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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