【作者】 吴纲；

【导师】 史俊南； 金岩；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2008， 博士

【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。更多还原

【Abstract】 Morphogenesis and development of tooth initiate from epithelial-mesenchymal interactions,indicating that stem cells of both epithelial and mesenchymal origin should be considered in stem cell based tooth regeneration using tissue engineering approach.Previous studies have demonstrated that tooth germ cells,dental pulp stem cells from adult or deciduous tooth and stem cells from apical papilla have the potential to differentiate into odontoblast lineages and perform the dentinogenesis.However, these cells are very limited and some of them can not be directly isolated from patients themselves in clinical practice.Bone marrow is the only source for non-dental stem cell which can demonstrate odontogenic potential with the induction of oral epithelium from E10 fetal mouse.In addition,the lack of seeding cells for enamel regeneration is always a big rock on the way of tooth regeneration.Recent studies highlighted in stem cells from hair follicle because of their tremendous plasticities and easier accessibility.In this paper,we explored the odontogenic potential of hair-follicle-derived stem cells and main results were listed as follows.PartⅠ.Culturing of hair follicle dermal stem cells and evaluation of their multiple differentiation capacity.Both cultured hair follicle dermal sheath cells and dermal papilla cells demonstrated a larger CFU efficiency and higher reproductive activity compared with interfollicular dermal fibroblasts.Follicle dermal stem cells were heterogeneous and lacking specific surface markers.However,directed differentiation potential of stem cells could be detected with heterogeneous cells which containing stem cells.We found that cultured follicle dermal cells including putative MSCs from aged GFP transgenic mice still presented plasticity, as indicated by the results of multiple differentiation assays.Stem cells from follicle dermal papilla presented a higher ALP activity than those from dermal sheath under the same osteogenic microenvironment.PartⅡ.Odontogenic potential of mesenchymal stem cells from hair follicle dermal papilla.In the in vitro mixed co-culture system containing apical bud cells,GFP labeled follicle dermal papilla cells and dental mesenchymal cells,DSP co-localized with GFP,indicating that some GFP~+ DPCs can differentiate to odontoblasts.These results also suggested that odontogenic microenvironment for non-dental stem cells could be obtained from outside of embryo. Tooth-germ-cell conditioned medium produced by apical bud cells and dental mesenchymal cells could also change the morphology and cell cycle of DPC and further drive these cells to express Dspp and Dmpl genes.PartⅢ.Tissue engineering a pulp-dentin complex using hair follicle dermal papilla cells.We combined an untreated dermal papilla cell pellet with a scaffold which preliminary binded with TGC-CM.Recombinants were cultured under subcutaneous condition of nude mouse.Histological analyses of recoverd explants showed that there was only a small quantity of homogeneous hard tissue instead of typical tubler dentin.Cell pellet of dermal papilla cells treated with TGC-CM preliminary could only form a bone like tissue after cultured in renal capsule for 3 weeks.These results suggested that terminal differentiation of odontoblast is a complicated process which could not accomplished with signaling molecules only.PartⅣ.Possibilty of enamel regeration using hair follicle epithelial stem cells.Outer root-sheath cells were cultured in vitro and demonstrated some typical characteristics of epithelial stem cells.These cells presented a larger CFU efficiency and higher reproductive activity compared with keratinocytes.Keratin 19 and integrinβ1 could be detected in these cells at the same time.We then recombined these cells with dental mesenchymes from first lower molar of E17 SD rats and apical papilla of 7 dpn C57BL/6 GFP transgenic mice.Epithelial cells from the back of E17 SD rats were used as control groups.Results showed that both dental mesenchymes could initiate the ameloblast differentiation of epithelial cells from the back of E17 SD rats.But there were no sufficient experimental evidence showed that hair follicle epithelial stem cells could differentiate to ameloblasts.In summary,our data showed for the first time that mesenchymal stem cells from hair follicle dermal papilla could differentiate to odontoblasts with the induction of dental epithelium from apical bud.Dental mesenchyme from apical papilla of 7 dpn C57BL/6 GFP transgenic mice as well as that from first lower molar of E 17 SD rat could initiate the ameloblast differentiation of epithelial cells from the back of E17 SD rats.We had not found the way to make hair follicle epithelial stem cells differentiate to ameloblasts yet.更多还原