毛囊源性干细胞成牙能力的实验研究

发布时间：2018-03-10 09:07

本文选题：形态发生　切入点：上皮-间充质相互作用　出处：《第四军医大学》2008年博士论文　论文类型：学位论文

【摘要】： 牙齿的发生和发育是在上皮-间充质相互作用的精确调控下完成的,这提示我们在选择种子细胞的时候必须同时考虑两种细胞,即上皮细胞和间充质细胞。目前发现的有望应用于牙组织工程的细胞大部分源于牙本身,如牙髓干细胞、脱落乳牙牙髓干细胞、牙周膜干细胞、根尖牙乳头干细胞等。在非牙源性成体干细胞方面,目前仅发现骨髓来源的细胞具有成牙潜能,而且必须依靠胚胎期的诱导条件才能分化为牙齿形成细胞。目前利用非牙源性细胞实现牙齿再生面临着种子细胞匮乏、诱导条件短缺的困难,另外,在成体中寻找适用于釉质再生的干细胞一直都是牙齿再生的瓶颈问题。毛囊是近年来干细胞研究的热点,毛囊中包含上皮和间充质两种来源的干细胞。本研究从成体干细胞可塑性和干细胞定向分化微环境两个方面入手,探讨利用毛囊来源的干细胞进行牙齿再生的可能性。 1毛囊真皮细胞的分离培养和干细胞生物学特性的研究采用酶消化联合机械分离的方法在体外成功培养了具有较强增殖能力、克隆形成能力的毛囊真皮鞘细胞和毛乳头细胞。通过对其可塑性的研究证实这两类细胞中包含具有多向分化能力的干细胞。但是这些干细胞具有异质性,缺乏特异性表面标志物,难以有效分选。而含有干细胞的混杂细胞可以用来进行干细胞定向分化研究,未经纯化的毛囊真皮细胞在成脂、成骨诱导条件下,其中的所包含的干细胞表现出成脂和成骨潜能。在相同的成骨诱导条件下,毛乳头细胞的碱性磷酸酶活性比真皮鞘细胞更强,因此可能更适用于硬组织再生。 2毛乳头间充质干细胞分化为成牙本质细胞在出生后小鼠下颌切牙根尖蕾牙胚细胞与毛乳头间充质干细胞混合共培养体系中,来自根尖蕾上皮的成牙信号能够促使毛乳头间充质干细胞分化为成牙本质细胞,说明诱导非牙源性干细胞成牙的微环境可以脱离发育早期牙胚的限制。在根尖蕾牙胚细胞条件培养液诱导下,毛乳头间充质细胞的形态和细胞周期明显改变并表达Dspp和Dmp1基因,说明来自根尖蕾细胞的上皮信号也能启动毛乳头间充质细胞的牙向分化。 3毛乳头间充质干细胞构建牙本质牙髓复合体的实验研究为了研究利用非牙源性干细胞在脱离牙胚组织的条件下构建组织工程牙齿的可能性,我们将把经过根尖蕾牙胚细胞条件培养液诱导的毛乳头间充质细胞团进行体内移植后,仅形成骨样组织。将根尖蕾牙胚细胞条件培养液结合到脱蛋白牙本质片制成的支架材料上,种子细胞以细胞团的形式与支架材料进行复合后体内移植。取材结果显示:在结合在支架材料上的条件培养基的作用下,对照组的牙髓干细胞可以分化为成牙本质细胞,规则地排列在支架材料表面,并且形成管状牙本质。而毛乳头细胞只在支架材料表面形成少量无定形基质。这一结果也说明仅仅依靠信号分子的作用,不能实现非牙源性细胞分化为成熟的成牙本质细胞。 4毛囊表皮干细胞应用于釉质再生的可能性利用组织块法成功培养毛囊上段的外根鞘细胞,这些细胞的自我更新能力强,呈克隆样生长,表达表皮干细胞的表面标志物β1整合素和角蛋白19,具有典型的表皮干细胞特征。在此基础上,我们利用E17胎鼠下颌第一磨牙牙乳头和出生后7 d C57BL/6 GFP小鼠下颌切牙根尖蕾间充质,分别与E17胎鼠表皮和体外培养的毛囊表皮干细胞进行重组,结果显示,E17胎鼠下颌第一磨牙牙乳头与生后7 d C57BL/6 GFP小鼠下颌切牙根尖蕾间充质均具有诱导E17胎鼠表皮细胞分化为成釉细胞的能力,但是没有充分的证据表明体外培养的毛囊表皮干细胞在这两种诱导条件下可以分化为成釉细胞。综上所述,本研究首次证实了毛乳头间充质干细胞在小鼠下颌切牙根尖蕾上皮信号的诱导下能够分化为成牙本质细胞,但是仍无法在脱离牙胚组织的条件下独立成牙。根尖蕾间充质对非牙源性上皮的诱导能力与E17下颌第一磨牙牙乳头相似,本实验暂无直接证据可以证明新生鼠触须部毛囊表皮干细胞具有分化为成釉细胞的潜能。
[Abstract]:The occurrence and development of teeth is accurate in regulation of epithelial mesenchymal interactions to complete, suggesting that we also consider two types of cells in the selection of seed cells to that of epithelial cells and mesenchymal cells. The potential application in dental tissue engineering cells mainly originates from the tooth itself, such as dental pulp stem cells, exfoliated cells of stem, periodontal ligament stem cells, apical papilla stem cells. In non dental stem cells into the body, currently only found bone marrow-derived cells have odontogenic potential, and must rely on embryonic induction conditions can differentiate into teeth forming cells for tooth regeneration using at present. Nonodontogenic cells facing seed cells induced by lack of conditions a shortage of, in addition, in adult looking for suitable for enamel regeneration stem cells have been the bottleneck problem of tooth regeneration is the hair follicle. In recent years, the focus of stem cell research is hair follicle, which contains two sources of epithelial and mesenchymal stem cells. In this study, from the two aspects of adult stem cell plasticity and stem cell oriented differentiation microenvironment, we explore the possibility of tooth regeneration using hair follicle stem cells.
Study on the isolation and culture of 1 hair follicle dermal cells and the biological characteristics of stem cells
In vitro culture has strong proliferation ability by using the method of enzyme digestion combined with mechanical isolation, clone forming ability of hair follicle dermal sheath cells and dermal papilla cells. Through the study of the plasticity of that contains stem cells capable of multi-directional differentiation of these two kinds of cells. But these stem cells are heterogeneous and lack of special specific surface markers and cell sorting. It is difficult to effectively mix containing stem cells can be used for the study of stem cell differentiation, without hair follicle dermal cells purified in adipogenic, osteogenic induction condition, which contains stem cells showed adipogenic and osteogenic potential of bone in the same. Under the induction of dermal papilla cells alkaline phosphatase activity than the dermal sheath cells is stronger, and therefore may be more suitable for hard tissue regeneration.
2 dermal papilla mesenchymal stem cells differentiate into odontoblast cells
In the apical bud of tooth germ cells and dermal papilla mesenchymal stem cells co culture system of mouse mandibular incisor teeth after birth, into a signal from the apical bud epithelium can promote dermal papilla mesenchymal stem cells differentiate into odontoblast cells, indicating that the micro environment induced by non dental stem cells into teeth can be separated from the development of early tooth germ in the apical bud. Cultured tooth germ cell conditioned medium induced by dermal papilla mesenchymal cell morphology and cell cycle changes and expression of Dspp and Dmp1 gene, indicating epithelial signals from apical bud cells can also initiate dermal papilla mesenchymal cells Odontogenetic differentiation.
An experimental study on the construction of dentin pulp complex of 3 dermal papilla mesenchymal stem cells
In order to study the possibility of cells for tissue engineering of teeth in the tooth germ tissue from under the condition of non dental stem, we will put through the apical bud tooth germ cell conditioned medium induced dermal papilla mesenchymal cells transplantation in vivo, only the formation of bone like tissues. The apical bud tooth germ cell conditioned medium according to deproteinized dentin pieces made of scaffolds, seed cells in vivo after compound in the form of cells and scaffold materials. The results showed that: in combination based on scaffolds of the conditioned medium under the action of the group of dental pulp stem cells can differentiate into odontoblasts were arranged regularly on the surface of the scaffold, and the formation of tubular dentin. While dermal papilla cells only in the surface of the scaffold to form a small amount of amorphous matrix. This result also shows that relying solely on the role of signal molecule, can't Non odontogenic cells are differentiated into mature odontoblast cells.
The possibility of application of 4 hair follicle epidermal stem cells to the regeneration of enamel
The outer root sheath hair follicle cells were cultured successfully by the method of tissue, the cell self-renewal ability, a clone like growth, expression of epidermal stem cell surface marker of integrin beta 1 and keratin 19, has the typical characteristics of epidermal stem cells. On this basis, we use E17 fetal rat mandibular first molars the dental papilla and 7 d after birth C57BL/6 GFP mouse mandibular apical bud mesenchyme, epidermal hair follicles were cultured with E17 in vitro and epidermal stem cells of fetal rat recombinant, results showed that E17 fetal rat mandibular first molar papilla and 7 d after birth C57BL/6 GFP mouse mandibular apical bud mesenchyme were with the induction of epidermal cell differentiation ability for E17 rat ameloblasts, but there is no evidence to show that these two kinds of cells in inducing conditions can differentiate into ameloblasts of epidermal hair follicle in vitro.
In summary, this study first confirmed that can differentiate into odontoblast like cells induced by dermal papilla mesenchymal stem cell apical bud in mouse mandibular epithelial signals, but is not from tooth germ tissue under the condition of independent teeth. The apical bud inducing ability between E17 and mandibular first molar dental papilla mesenchymal of nonodontogenic on skin is similar, no direct evidence of this experiment can prove that the newborn rat whisker hair follicle epidermal stem cells can differentiate into ameloblasts potential.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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