诱导人表皮干细胞牙向分化的研究
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摘要
许多外胚层器官,如牙齿、毛发等均源于上皮组织和间充质组织。在牙胚发育过程中,上皮-间充质之间的相互作用调控其形态的发生和细胞的分化。由此推论,利用干细胞进行的牙齿器官的再生也需要上皮源性和间充质源性的干细胞的参与。本论文的目的是探讨人表皮干细胞的成牙能力,摸索适合牙齿再生的人上皮源性细胞及再生条件,为牙齿再生的组织工程找出种子细胞和方法依据。在本论文的研究中,我们分离、培养和鉴定了人的表皮干细胞,并将培养的表皮膜片结构与小鼠E13.5的具有诱导牙齿发育能力的牙胚间充质重组。重组后的嵌合体在裸鼠囊膜下能发育形成牙齿的结构,具有发育良好的牙本质;但在形态学上表皮干细胞却未分化为成熟的成釉细胞,形成的牙齿缺乏釉质。为了进一步建立和完善牙齿再生过程中表皮干细胞作为牙上皮的技术,我们往嵌合体中分别添加外源生长因子FGF8、Shh、BMP4。结果发现添加FGF8生长因子的嵌合体具完整的牙冠结构(牙髓、牙本质、牙釉质以及成釉细胞);添加Shh生长因子的嵌合体跟没有添加因子的没有明显区别;添加BMP4生长因子的嵌合体骨化明显。这表明由人表皮干细胞构成的上皮组织可以支持牙齿的发育,添加上皮有关的生长因子FGF8有助于表皮干细胞牙向分化。
Many ectodermal organs, such as teeth and feathers, all develop from epithelium and mesenchyme. The reciprocal interactions of epithelium-mesenchyme are regulators of morphogenesis and cell differentiation in the process of teeth growth. Therefore, we can deduce that the regeneration of tooth organs based on stem cells requires participation of stem cells derived from epithelium or mesenchyme. objective of this study is explore the potentail capacity of human keratinocyte stem cells for odontogenetic differetiaontion in order to find suitable human epithelium-original stem cells and regeneration conditions for tooth regeneration, and an appropriate kind of cell and regenerative model for tissue engineering of tooth regeneration. In our research, hKSCs were isolated、cultured and identified. We integrate hKSCs into mouse tooth germs mesenchymal with odontogenic potential at Embryonic day 13.5(E13.5) as recombinant, then transplant the recombinant to immuno-compromised mice sub-renal culture to find out if hKSCs can acquire the odontogenic competence and participate in the tooth development.Result of the recombinant culture shows that the recombinated tooth can develop into dental structures and possessed well-developed dentin; but hKSCs haven't induced into ameloblasts in morphologyo In order to fatherly establish and improve the technology of hKSCs as dental epithelial in the process of tooth regeneratin, we appended FGF8、Shh、BMP4 into the recombinated chimeric respectively. The results shows that the recombinated tooth contained FGF8 growth factor possesses complete tooth coronal structure (dental pulp、dentiiu enamel、ameloblasts).;the one appended with Shh is similar to chimeric without growth factor, and the one with BMP4 has distinct ossification .We conclude that hKSCs can support dental development as a role of epithelial; and appended FGF8 induce additionally facilitate they odontogenic differentiation.
Many ectodermal organs, such as teeth and feathers, all develop from epithelium and mesenchyme. The reciprocal interactions of epithelium-mesenchyme are regulators of morphogenesis and cell differentiation in the process of teeth growth. Therefore, we can deduce that the regeneration of tooth organs based on stem cells requires participation of stem cells derived from epithelium or mesenchyme. objective of this study is explore the potentail capacity of human keratinocyte stem cells for odontogenetic differetiaontion in order to find suitable human epithelium-original stem cells and regeneration conditions for tooth regeneration, and an appropriate kind of cell and regenerative model for tissue engineering of tooth regeneration. In our research, hKSCs were isolated、cultured and identified. We integrate hKSCs into mouse tooth germs mesenchymal with odontogenic potential at Embryonic day 13.5(E13.5) as recombinant, then transplant the recombinant to immuno-compromised mice sub-renal culture to find out if hKSCs can acquire the odontogenic competence and participate in the tooth development.Result of the recombinant culture shows that the recombinated tooth can develop into dental structures and possessed well-developed dentin; but hKSCs haven't induced into ameloblasts in morphologyo In order to fatherly establish and improve the technology of hKSCs as dental epithelial in the process of tooth regeneratin, we appended FGF8、Shh、BMP4 into the recombinated chimeric respectively. The results shows that the recombinated tooth contained FGF8 growth factor possesses complete tooth coronal structure (dental pulp、dentiiu enamel、ameloblasts).;the one appended with Shh is similar to chimeric without growth factor, and the one with BMP4 has distinct ossification .We conclude that hKSCs can support dental development as a role of epithelial; and appended FGF8 induce additionally facilitate they odontogenic differentiation.
引文
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