【摘要】： 在組織工程研究領(lǐng)域中,牙齒再生技術(shù)的建立已成為該領(lǐng)域的熱點(diǎn)。由于牙齒發(fā)育模式的獨(dú)特性和結(jié)構(gòu)相對簡單,牙齒再生將會成為可能。牙齒形態(tài)的發(fā)生和分化,需要上皮來源的細(xì)胞和神經(jīng)嵴來源的間充質(zhì)細(xì)胞相互誘導(dǎo),同樣誘導(dǎo)干細(xì)胞再生牙齒也需要上皮源性和間充質(zhì)源性的細(xì)胞參與。目前間充質(zhì)來源的干細(xì)胞很容易獲得,像牙髓干細(xì)胞,骨髓干細(xì)胞都可以被誘導(dǎo)為類牙本質(zhì)的結(jié)構(gòu),而胚胎干細(xì)胞誘導(dǎo)效率低,獲得比較困難,又面臨著倫理問題,所以目前在組織工程領(lǐng)域中,成體干細(xì)胞成為該領(lǐng)域的研究趨勢。本論文的目的是想建立表皮干細(xì)胞的分離和培養(yǎng)技術(shù)體系,開展誘導(dǎo)人類表皮干細(xì)胞制備牙上皮的研究工作。在本實(shí)驗(yàn)的研究中,建立了比較完善的表皮干細(xì)胞培養(yǎng)體系,并且得出在成纖維培養(yǎng)條件下,有利于表皮干細(xì)胞的擴(kuò)增和表型的維持;在FGF8生長因子誘導(dǎo)條件下,表皮膜片與E13.5的牙間充質(zhì)細(xì)胞重組,重組后的嵌合體移植到裸鼠腎囊膜三周,組織學(xué)切片,發(fā)現(xiàn)表皮干細(xì)胞可以被誘導(dǎo)分化為成釉細(xì)胞,并且成釉細(xì)胞分泌牙釉質(zhì);為了檢測嵌合體牙齒中成釉細(xì)胞的功能性,本實(shí)驗(yàn)檢測特異性識別人抗體的Ameloblastin,MMP-20,FGF8,結(jié)果說明FGF8生長因子可以誘導(dǎo)表皮干細(xì)胞形成有功能的成釉細(xì)胞。在實(shí)驗(yàn)過程中,發(fā)現(xiàn)老鼠牙胚在帽狀早期FGF8開始不表達(dá),而人類牙齒從早期到分泌期FGF8都有表達(dá)。而在嵌合體牙齒中,FGF8表達(dá)很強(qiáng)烈,說明再生出的牙齒趨向于人類牙齒。同時(shí)本實(shí)驗(yàn)用DSP抗體檢測嵌合體牙齒中成牙本質(zhì)細(xì)胞的功能,發(fā)現(xiàn)其發(fā)分泌的牙本質(zhì)DSP有強(qiáng)烈的表達(dá)。實(shí)驗(yàn)過程中,由于成牙率很低,想通過長時(shí)期表達(dá)FGF8這種生長因子來提高成牙率,所以本實(shí)驗(yàn)又構(gòu)建了長時(shí)期表達(dá)FGF8生長因子的慢病毒lentiviruse-mfgf8,結(jié)果嵌合體也可以成牙,并且表皮干細(xì)胞可以被誘導(dǎo)為成釉細(xì)胞,雖然成牙率有提高,但是實(shí)驗(yàn)次數(shù)比較少(兩粒有一粒成牙),對于這一結(jié)果有待于進(jìn)一步研究�？傊掀碓吹谋砥じ杉�(xì)胞可以支持牙齒再生,并且在FGF8的誘導(dǎo)下,可以分化成成釉細(xì)胞,分泌釉質(zhì)。這一結(jié)果將為牙齒再生的臨床實(shí)踐奠定理論和技術(shù)基礎(chǔ),對于干細(xì)胞在組織工程研究領(lǐng)域中的應(yīng)用研究有普遍意義。
[Abstract]:In the field of tissue engineering, the establishment of tooth regeneration technology has become a hot spot in this field. Tooth regeneration will be possible because of the unique and relatively simple structure of the dental development model. Tooth morphogenesis and differentiation need to be induced by epithelial-derived cells and neural crister-derived mesenchymal cells as well as epithelial and mesenchymal cells to induce tooth regeneration from stem cells. At present, mesenchymal stem cells are very easy to obtain, such as dental pulp stem cells, bone marrow stem cells can be induced into dentin-like structure, while embryonic stem cells are less efficient, more difficult to obtain, and face ethical problems. Therefore, in the field of tissue engineering, adult stem cells have become the research trend in this field. The purpose of this thesis is to establish the technical system of isolation and culture of epidermal stem cells and to develop the research of inducing human epidermal stem cells to prepare dental epithelium. In this study, a more perfect culture system of epidermal stem cells was established, and the results showed that under the condition of fibroblast culture, it was beneficial to the expansion of epidermal stem cells and the maintenance of phenotypes. Under the condition of FGF8 growth factor induction, epidermal membrane and E13.5 dental mesenchymal cells were recombined and transplanted into the renal capsule membrane of nude mice for three weeks. Histological sections showed that epidermal stem cells could be induced to differentiate into ameloblast. And enamel cells secreted enamel. In order to detect the function of ameloblasts in chimeric teeth, the Ameloblastin,MMP-20,FGF8, results showed that FGF8 growth factor could induce epidermal stem cells to form functional ameloblasts. During the experiment, it was found that mouse tooth germ did not express FGF8 at the early stage of capping, but FGF8 was expressed in human teeth from early stage to secretory stage. In chimeric teeth, FGF8 is strongly expressed, suggesting that regenerated teeth tend to be human teeth. At the same time, DSP antibody was used to detect the function of odontoblast in chimeric teeth, and it was found that there was strong expression of dentin DSP in chimeric teeth. In the course of the experiment, because of the low rate of tooth formation, we want to increase the rate of tooth formation by expressing FGF8 for a long time, so we also constructed the lentivirus lentiviruse-mfgf8, result chimera which expressed FGF8 growth factor for a long time. Epidermal stem cells can be induced as ameloblasts, although the rate of tooth formation is increased, but the number of experiments is relatively small (two grains have one tooth), this result needs further study. In conclusion, epidermal stem cells derived from epithelium can support tooth regeneration, and can differentiate into ameloblasts and secrete enamel under the induction of FGF8. The results will lay a theoretical and technical foundation for the clinical practice of tooth regeneration, and will be of general significance for the application of stem cells in tissue engineering.
【學(xué)位授予單位】：福建師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 呂淑坤;;表皮干細(xì)胞在組織工程創(chuàng)面修復(fù)中的應(yīng)用[J];中國組織工程研究;2012年27期

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本文編號：2375444