【作者】 杜生荣；

【导师】 张彦定；

【作者基本信息】 福建师范大学 ， 发育生物学， 2009， 硕士

【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。更多还原

【Abstract】 In the field of tissue engineering research, the technology of tooth regeneration has become a hot field. Because of unique pattern of tooth development and relatively simple structure, tooth regeneration will be possible. Sequential and reciprocal interactions between the cranial neural crest-derived mesenchymal cells and the stomadial epithelium regulate tooth morphogenesis and differentiation.so regeneration of tooth based on stem cells needs participation of epithelium-derived and mesenchyme-derived. At present,mesenchymal stem cells are got easily,such as dental pulp stem cell and bone marrow stem cells can be induced into dentin-like structure;But Embryonic stem cell ,low inducd efficiency, got difficult and faced ethical issues,so at present , research of Adult stem cells has been advanced in the field of tissue engineering research.The purpose of paper focuse on isolation and culture technique of epidermal stem cells , And carry out inducing human epidermal stem cell research .In this essay,we have established a relatively perfect system of culture of epidermal stem cells, amplification and maintenance of phenotypic of epidermal stem cells can be more effective in human fibroblast conditioned medium.we show that epithelial sheets of cultured human keratinocytes,when confronted with mouse E13.5 embryonic dental mesenchyme and in the presence of FGF8,which were subsequently subjected to subrenal culture for 4-week.differentiated into enamel-secreted ameloblasts and formed a humanmouse chimeric whole tooth crown.In order to detect function of ameloblasts of humanmouse chimeric tooth,immunostaining assys showed specific presence of ameloblastin ,MMP-20 and FGF8,specific markers for differentiated ameloblasts,in the elongated and the enamel.Our result demonstrate that human epidermal stem cells are capable of differentiating into ameloblasts and producing enamel.In the experiment ,we found that bud-like early of mouse tooth dose not express fgf8 ,but human teeth have the expression of fgf8 from beginning phase to secretory phase. Human-mouse chimeric tooth express fgf8 strongly, so regenerational teeth tend to human teeth.At the same time,we detect odontoblastic function with DSP antibody, found that specific presence of DSP antibody in dentin.But in our experiments,formational rate of chimeric tooth is low, so we construct lentiviruse-mfgf8 to improve formational rate .As a result,chimeras can form tooth,with that epidermal stem cells can different into ameloblasts and dental formational rate have been improved.But as relatively small number of experiments (one tooth of two chimeric tissues) ,for this result we need continue to study.In a word, epithelial sheets of cultured human epidermal stem cell ,when confronted with mouse embryonic dental mesenchyme and in the presence of FGF8,differentiated into enamel-secreted ameloblasts and formed a human-mouse chimeric whole tooth crown.This result will lay the theoretical and technical basis for dental regeneration, it has universal significance in tissue engineering of stem cells.更多还原