【作者】 焦鹏；

【导师】 张彦定；

【作者基本信息】 福建师范大学 ， 发育生物学， 2005， 硕士

【摘要】 本研究尝试建立人类乳牙牙髓干细胞（stem cells from humanexfoliated deciduous teeth,SHED）体外快速扩增方法并对利用SHED进行牙齿再生进行了初步的探索。主要研究工作包括:SHED的分离培养及初步鉴定,SHED体外快速扩增方法的建立,Alu和Pf1地高辛标记探针的制备, E10.5鼠胚（小白鼠胚胎发育第10.5天）第一腮弓上皮和第二腮弓上皮分别与SHED的重组,SHED以50%比例掺入到E13.5鼠胚牙间充质细胞后与E13.5鼠胚牙上皮的重组,以及DNA原位杂交检测鼠源组织和人源组织。 实验结果表明,通过分离培养SHED,确定用低密度(1.5cells/cm²)接种更有利于SHED的体外快速扩增,通过低密度接种连续传代三次也就是大约六周的时间里,SHED大约扩增了10^1O—10¹¹倍。通过克隆形成效率实验与矿化结节实验,确定这些扩增后的细胞仍然具有与第一次进行低密度扩增时的SHED即起始时的SHED基本相同的克隆形成能力而且在体外仍能形成矿化结节,因此保留了多分化潜能。利用小白鼠为模型建立了一套牙齿异体再生技术并正在不断完善,通过嵌合体组织重组实验,不但证明了不同种间的组织可以相互作用,正常的发育成牙齿结构,而且初步证实在牙齿的再生过程中SHED发育形成牙组织的潜能是以被诱导者的形式存在,即SHED只具有应答能力,而不具有诱导牙齿发育的潜能。作为被诱导者,对具有诱导能力的上皮组织或问充质组织作出应答反应,共同发育形成牙齿的结构。更多还原

【Abstract】 This study attempted to establish the method of rapid expansion of stem cells from human exfoliated deciduous teeth(SHED) and did the elementary exploration about regeneration of tooth from SHED. The studies included isolating, culturing and identifying of SHED; establishing the method of rapid expansion of SHED; Alu and Pfl digoxigenin-probe preparative; recombinating between the first branchial arch epitheliums of mouse embryos (E10.5) and SHED; recombinating between the second branchial arch epitheliums of mouse embryos (E10.5) and SHED; recombinating between tooth epitheliums of mouse embryos (E13.5) and tooth mesenchyme cells of mouse embryos (E13.5) mixed into 50% SHE D; checking mouse tissue and human tissue with DNA in situ hybridization.By isolating and culturing SHED , the result demonstrated that the cells grew most rapidly when they were initially plated at low densities (1.5 cells/cm2). In three passages in which the cells were plated at low density, they were amplified about 10~10-10~11-fold in 6 weeks. The cells retained their ability to form calcified deposition and the average percent cfus at the end of the third passage(six weeks) was about the same as the initial value for the samples (55% vs. 58%) and therefore apparently retained their multipotentiality for differentiation. This study used mouse as an animal model to establish a set of technique and we are improving it. By using it, mouse tooth can be regenerated in ectopic body. Human and mouse chimera tissue-recombination experiment not only demonstrated that the reciprocal interactions between tissues of different species may induce tooth development but also demonstrated elementarily that SHED only haveodontogenic competence and they have not odontogenic potential. SHED can be induced by epitheliums or mesenchyme cells that both have odontogenic potential to differentiate into tooth tissue.更多还原