本文選題：形態(tài)發(fā)生　切入點(diǎn)：上皮-間充質(zhì)相互作用　出處：《第四軍醫(yī)大學(xué)》2008年博士論文　論文類型：學(xué)位論文

【摘要】： 牙齒的發(fā)生和發(fā)育是在上皮-間充質(zhì)相互作用的精確調(diào)控下完成的,這提示我們?cè)谶x擇種子細(xì)胞的時(shí)候必須同時(shí)考慮兩種細(xì)胞,即上皮細(xì)胞和間充質(zhì)細(xì)胞。目前發(fā)現(xiàn)的有望應(yīng)用于牙組織工程的細(xì)胞大部分源于牙本身,如牙髓干細(xì)胞、脫落乳牙牙髓干細(xì)胞、牙周膜干細(xì)胞、根尖牙乳頭干細(xì)胞等。在非牙源性成體干細(xì)胞方面,目前僅發(fā)現(xiàn)骨髓來(lái)源的細(xì)胞具有成牙潛能,而且必須依靠胚胎期的誘導(dǎo)條件才能分化為牙齒形成細(xì)胞。目前利用非牙源性細(xì)胞實(shí)現(xiàn)牙齒再生面臨著種子細(xì)胞匱乏、誘導(dǎo)條件短缺的困難,另外,在成體中尋找適用于釉質(zhì)再生的干細(xì)胞一直都是牙齒再生的瓶頸問(wèn)題。毛囊是近年來(lái)干細(xì)胞研究的熱點(diǎn),毛囊中包含上皮和間充質(zhì)兩種來(lái)源的干細(xì)胞。本研究從成體干細(xì)胞可塑性和干細(xì)胞定向分化微環(huán)境兩個(gè)方面入手,探討利用毛囊來(lái)源的干細(xì)胞進(jìn)行牙齒再生的可能性。 1毛囊真皮細(xì)胞的分離培養(yǎng)和干細(xì)胞生物學(xué)特性的研究 采用酶消化聯(lián)合機(jī)械分離的方法在體外成功培養(yǎng)了具有較強(qiáng)增殖能力、克隆形成能力的毛囊真皮鞘細(xì)胞和毛乳頭細(xì)胞。通過(guò)對(duì)其可塑性的研究證實(shí)這兩類細(xì)胞中包含具有多向分化能力的干細(xì)胞。但是這些干細(xì)胞具有異質(zhì)性,缺乏特異性表面標(biāo)志物,難以有效分選。而含有干細(xì)胞的混雜細(xì)胞可以用來(lái)進(jìn)行干細(xì)胞定向分化研究,未經(jīng)純化的毛囊真皮細(xì)胞在成脂、成骨誘導(dǎo)條件下,其中的所包含的干細(xì)胞表現(xiàn)出成脂和成骨潛能。在相同的成骨誘導(dǎo)條件下,毛乳頭細(xì)胞的堿性磷酸酶活性比真皮鞘細(xì)胞更強(qiáng),因此可能更適用于硬組織再生。 2毛乳頭間充質(zhì)干細(xì)胞分化為成牙本質(zhì)細(xì)胞 在出生后小鼠下頜切牙根尖蕾牙胚細(xì)胞與毛乳頭間充質(zhì)干細(xì)胞混合共培養(yǎng)體系中,來(lái)自根尖蕾上皮的成牙信號(hào)能夠促使毛乳頭間充質(zhì)干細(xì)胞分化為成牙本質(zhì)細(xì)胞,說(shuō)明誘導(dǎo)非牙源性干細(xì)胞成牙的微環(huán)境可以脫離發(fā)育早期牙胚的限制。在根尖蕾牙胚細(xì)胞條件培養(yǎng)液誘導(dǎo)下,毛乳頭間充質(zhì)細(xì)胞的形態(tài)和細(xì)胞周期明顯改變并表達(dá)Dspp和Dmp1基因,說(shuō)明來(lái)自根尖蕾細(xì)胞的上皮信號(hào)也能啟動(dòng)毛乳頭間充質(zhì)細(xì)胞的牙向分化。 3毛乳頭間充質(zhì)干細(xì)胞構(gòu)建牙本質(zhì)牙髓復(fù)合體的實(shí)驗(yàn)研究 為了研究利用非牙源性干細(xì)胞在脫離牙胚組織的條件下構(gòu)建組織工程牙齒的可能性,我們將把經(jīng)過(guò)根尖蕾牙胚細(xì)胞條件培養(yǎng)液誘導(dǎo)的毛乳頭間充質(zhì)細(xì)胞團(tuán)進(jìn)行體內(nèi)移植后,僅形成骨樣組織。將根尖蕾牙胚細(xì)胞條件培養(yǎng)液結(jié)合到脫蛋白牙本質(zhì)片制成的支架材料上,種子細(xì)胞以細(xì)胞團(tuán)的形式與支架材料進(jìn)行復(fù)合后體內(nèi)移植。取材結(jié)果顯示:在結(jié)合在支架材料上的條件培養(yǎng)基的作用下,對(duì)照組的牙髓干細(xì)胞可以分化為成牙本質(zhì)細(xì)胞,規(guī)則地排列在支架材料表面,并且形成管狀牙本質(zhì)。而毛乳頭細(xì)胞只在支架材料表面形成少量無(wú)定形基質(zhì)。這一結(jié)果也說(shuō)明僅僅依靠信號(hào)分子的作用,不能實(shí)現(xiàn)非牙源性細(xì)胞分化為成熟的成牙本質(zhì)細(xì)胞。 4毛囊表皮干細(xì)胞應(yīng)用于釉質(zhì)再生的可能性 利用組織塊法成功培養(yǎng)毛囊上段的外根鞘細(xì)胞,這些細(xì)胞的自我更新能力強(qiáng),呈克隆樣生長(zhǎng),表達(dá)表皮干細(xì)胞的表面標(biāo)志物β1整合素和角蛋白19,具有典型的表皮干細(xì)胞特征。在此基礎(chǔ)上,我們利用E17胎鼠下頜第一磨牙牙乳頭和出生后7 d C57BL/6 GFP小鼠下頜切牙根尖蕾間充質(zhì),分別與E17胎鼠表皮和體外培養(yǎng)的毛囊表皮干細(xì)胞進(jìn)行重組,結(jié)果顯示,E17胎鼠下頜第一磨牙牙乳頭與生后7 d C57BL/6 GFP小鼠下頜切牙根尖蕾間充質(zhì)均具有誘導(dǎo)E17胎鼠表皮細(xì)胞分化為成釉細(xì)胞的能力,但是沒(méi)有充分的證據(jù)表明體外培養(yǎng)的毛囊表皮干細(xì)胞在這兩種誘導(dǎo)條件下可以分化為成釉細(xì)胞。 綜上所述,本研究首次證實(shí)了毛乳頭間充質(zhì)干細(xì)胞在小鼠下頜切牙根尖蕾上皮信號(hào)的誘導(dǎo)下能夠分化為成牙本質(zhì)細(xì)胞,但是仍無(wú)法在脫離牙胚組織的條件下獨(dú)立成牙。根尖蕾間充質(zhì)對(duì)非牙源性上皮的誘導(dǎo)能力與E17下頜第一磨牙牙乳頭相似,本實(shí)驗(yàn)暫無(wú)直接證據(jù)可以證明新生鼠觸須部毛囊表皮干細(xì)胞具有分化為成釉細(xì)胞的潛能。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

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本文編號(hào)：1592663