發(fā)布時(shí)間：2018-12-15 20:43

【摘要】：牙骨質(zhì)是覆蓋在牙根表面的特殊礦化組織，它借助牙周膜纖維將牙齒與牙槽骨相連，在維持牙齒結(jié)構(gòu)的穩(wěn)定和正常生理功能上發(fā)揮著重要作用。當(dāng)牙骨質(zhì)發(fā)生吸收或破壞時(shí)，其自我修復(fù)能力有限，原因在于隨著牙齒的發(fā)育完成，儲(chǔ)存在牙周組織中的成牙骨質(zhì)細(xì)胞數(shù)量有限同時(shí)也不易獲得。人牙周膜干細(xì)胞(Humanperiodontal ligament stem cells，hPDLSCs)具有自我增殖和多向分化潛能，是牙周組織工程的種子細(xì)胞。hPDLSCs在不同的培養(yǎng)環(huán)境和細(xì)胞因子的作用下，可不斷增殖分化成為成牙骨質(zhì)細(xì)胞、成骨細(xì)胞和成纖維細(xì)胞，修復(fù)牙周缺損，使牙周組織再生。目前已知的hPDLSCs向成牙骨質(zhì)細(xì)胞分化的轉(zhuǎn)錄信號(hào)通路有BMP(Bonemorphogenetic protein)和Wnt/β-catenin信號(hào)等。研究顯示BMP-2可促進(jìn)牙槽骨、牙骨質(zhì)和牙周韌帶的再生，也可以誘導(dǎo)成牙骨質(zhì)細(xì)胞的祖細(xì)胞（牙囊細(xì)胞）向成牙骨質(zhì)細(xì)胞方向分化,并刺激細(xì)胞表達(dá)牙骨質(zhì)標(biāo)志性蛋白牙骨質(zhì)附著蛋白(Cementumattachment protein, CAP)和/或牙骨質(zhì)蛋白(Cementum protein1, CEMP-1),本實(shí)驗(yàn)希望通過建立由牙胚細(xì)胞(Tooth germ cells, TGC)和BMP-2構(gòu)成的體外微環(huán)境，誘導(dǎo)hPDLSCs向成牙骨質(zhì)細(xì)胞分化，來探究牙骨質(zhì)的再生及發(fā)育機(jī)制。 實(shí)驗(yàn)方法 1. hPDLSCs的體外培養(yǎng)和鑒定 2. TGC誘導(dǎo)培養(yǎng)基的制作 3. TGC聯(lián)合BMP-2體外誘導(dǎo)hPDLSCs，，對(duì)誘導(dǎo)后的細(xì)胞進(jìn)行形態(tài)學(xué)觀察、茜素紅染色，堿性磷酸酶(Alkaline Phosphatase, ALP)活性檢測(cè)以及Real-time PCR檢測(cè)。 實(shí)驗(yàn)結(jié)果 1.利用免疫磁珠法篩選所得的hPDLSCs，間充質(zhì)干細(xì)胞標(biāo)志物STRO-1和血管周圍細(xì)胞標(biāo)志物CD146表達(dá)陽性，波形蛋白(Vimentin)表達(dá)陽性，角蛋白(Cytokeratin)表達(dá)陰性。 2.ALP活性檢測(cè)顯示，誘導(dǎo)組細(xì)胞ALP活性較對(duì)照組有顯著升高(P0.001)。 3. RT-PCR檢測(cè)顯示誘導(dǎo)組成牙骨質(zhì)細(xì)胞相關(guān)基因：ALP、CAP，CEMP-1及OCN的轉(zhuǎn)錄水平表達(dá)量增加(P0.001)。 4.茜素紅染色顯示，誘導(dǎo)組細(xì)胞表面出現(xiàn)大量礦化結(jié)節(jié)(P0.001)。 結(jié)論 TGC和BMP-2聯(lián)合作用能夠誘導(dǎo)hPDLSCs向成牙骨質(zhì)細(xì)胞的表型分化。
[Abstract]:Cementum is a special mineralized tissue covering the root surface. It connects teeth to alveolar bone with periodontal ligament fiber and plays an important role in maintaining the stability of tooth structure and normal physiological function. When the cementum is absorbed or destroyed, its self-repair ability is limited, because with the completion of tooth development, the number of cementoblasts stored in periodontal tissue is also limited and difficult to obtain. Human periodontal ligament stem cells (Humanperiodontal ligament stem cells,hPDLSCs) have the potential of self-proliferation and multidirectional differentiation and are seed cells of periodontal tissue engineering. HPDLSCs can proliferate and differentiate into cementum cells under different culture environments and cytokines. Osteoblasts and fibroblasts repair periodontal defects and make periodontal tissue regenerate. The known transcriptional signaling pathways for hPDLSCs differentiation into cementoblast include BMP (Bonemorphogenetic protein) and Wnt/ 尾-catenin. Studies have shown that BMP-2 can promote the regeneration of alveolar bone, cementum and periodontal ligament, and induce the differentiation of cementoblast progenitor cells (dental follicle cells) into cementoblast cells. And stimulate the expression of cementum iconic protein cementum attachment protein (Cementumattachment protein, CAP) and / or cementum protein (Cementum protein1, CEMP-1). The aim of this study was to establish a new method for the expression of cementum attachment protein (Tooth germ cells,) in dental germ cells. In order to explore the mechanism of cementum regeneration and development, hPDLSCs was induced to differentiate into cementoblast cells by in vitro microenvironment composed of TGC and BMP-2. Experimental method 1. In vitro culture and identification of hPDLSCs. The preparation of TGC induction medium. The morphological changes, alizarin red staining, alkaline phosphatase (Alkaline Phosphatase, ALP) activity and Real-time PCR detection were observed by TGC combined with hPDLSCs, induced by BMP-2 in vitro. Experimental results 1. The expression of hPDLSCs, mesenchymal stem cell markers STRO-1 and CD146, vimentin (Vimentin) and keratin (Cytokeratin) were positive by immunomagnetic beads. 2.ALP activity test showed that the ALP activity in the induced group was significantly higher than that in the control group (P0. 001). 3. RT-PCR analysis showed that the transcriptional levels of ALP,CAP,CEMP-1 and OCN were increased (P0.001). 4. Alizarin red staining showed that a large number of mineralized nodules appeared on the surface of cells in the induction group (P0.001). Conclusion the combined action of TGC and BMP-2 can induce the phenotypic differentiation of hPDLSCs into cementoblasts.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R781


本文編號(hào)：2381260