人表皮干细胞分化成成釉细胞的研究
发布时间：2018-12-12 23:34
【摘要】： 在组织工程研究领域中,牙齿再生技术的建立已成为该领域的热点。由于牙齿发育模式的独特性和结构相对简单,牙齿再生将会成为可能。牙齿形态的发生和分化,需要上皮来源的细胞和神经嵴来源的间充质细胞相互诱导,同样诱导干细胞再生牙齿也需要上皮源性和间充质源性的细胞参与。目前间充质来源的干细胞很容易获得,像牙髓干细胞,骨髓干细胞都可以被诱导为类牙本质的结构,而胚胎干细胞诱导效率低,获得比较困难,又面临着伦理问题,所以目前在组织工程领域中,成体干细胞成为该领域的研究趋势。本论文的目的是想建立表皮干细胞的分离和培养技术体系,开展诱导人类表皮干细胞制备牙上皮的研究工作。在本实验的研究中,建立了比较完善的表皮干细胞培养体系,并且得出在成纤维培养条件下,有利于表皮干细胞的扩增和表型的维持;在FGF8生长因子诱导条件下,表皮膜片与E13.5的牙间充质细胞重组,重组后的嵌合体移植到裸鼠肾囊膜三周,组织学切片,发现表皮干细胞可以被诱导分化为成釉细胞,并且成釉细胞分泌牙釉质;为了检测嵌合体牙齿中成釉细胞的功能性,本实验检测特异性识别人抗体的Ameloblastin,MMP-20,FGF8,结果说明FGF8生长因子可以诱导表皮干细胞形成有功能的成釉细胞。在实验过程中,发现老鼠牙胚在帽状早期FGF8开始不表达,而人类牙齿从早期到分泌期FGF8都有表达。而在嵌合体牙齿中,FGF8表达很强烈,说明再生出的牙齿趋向于人类牙齿。同时本实验用DSP抗体检测嵌合体牙齿中成牙本质细胞的功能,发现其发分泌的牙本质DSP有强烈的表达。实验过程中,由于成牙率很低,想通过长时期表达FGF8这种生长因子来提高成牙率,所以本实验又构建了长时期表达FGF8生长因子的慢病毒lentiviruse-mfgf8,结果嵌合体也可以成牙,并且表皮干细胞可以被诱导为成釉细胞,虽然成牙率有提高,但是实验次数比较少(两粒有一粒成牙),对于这一结果有待于进一步研究。总之上皮来源的表皮干细胞可以支持牙齿再生,并且在FGF8的诱导下,可以分化成成釉细胞,分泌釉质。这一结果将为牙齿再生的临床实践奠定理论和技术基础,对于干细胞在组织工程研究领域中的应用研究有普遍意义。
[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.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2375444
[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.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
【引证文献】
相关期刊论文 前1条
1 吕淑坤;;表皮干细胞在组织工程创面修复中的应用[J];中国组织工程研究;2012年27期
，本文编号：2375444
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