诱导小鼠胚胎干细胞向牙齿上皮样细胞分化的实验研究

发布时间：2018-06-27 15:36

本文选题：胚胎干细胞 + 牙源性上皮　；参考：《第四军医大学》2010年博士论文

【摘要】： 牙齿的发育过程是依赖于预定牙齿发生部位的上皮组织与其下方的间充质之间的相互诱导而发生的。因此,牙齿的生物性再生至少需要两种细胞:釉质形成所需的牙源性上皮细胞和形成牙髓牙本质复合体的牙源性间充质细胞。到目前为止,有关牙源性间充质细胞的替代研究较多,例如成体的牙髓干细胞、骨髓基质细胞、皮肤的真皮细胞以及毛囊细胞等均被证明具有向牙源性间充质细胞转化的能力。然而,在寻找合适的细胞向牙源性上皮细胞替代方面成功的研究较少。尽管有学者认为成体骨髓来源的细胞可以向成釉细胞转化,但是它可以同时向成釉细胞样细胞和牙源性间充质细胞这两种细胞转化的能力限制了它的应用。有研究在组织工程牙齿的构建中口腔腭粘膜上皮可以替代牙源性上皮,但是此研究必须建立在使用胚胎期或出生1天的自体口腔腭粘膜上皮的基础上,因此也限制了它的应用。这种局限性促使我们寻找一种新的细胞能够向牙源性上皮方向分化。胚胎干细胞具有自我更新和多向分化的能力,近年来受到许多学者广泛关注。有研究证实胚胎干细胞可以分化为皮肤表皮细胞,肺上皮细胞以及胸腺上皮细胞等,但是胚胎干细胞是否可以向牙源性上皮方向转化还尚未见报道。因此对于这一问题的回答,无疑于对解决牙齿再生研究中上皮源性种子细胞来源的瓶颈问题具有重要意义。本课题主要进行了以下几方面的研究: 1.胚胎干细胞的培养及鉴定目的:培养胚胎干细胞R1系及CGR8系并对其进行鉴定。方法:胚胎干细胞极易受外界因素的影响,对生长条件要求非常苛刻。常规的胚胎干细胞培养液中含有高糖MEM培养基、胎牛血清、L-谷氨酰胺、2-巯基乙醇和非必需氨基酸等,为了防止胚胎干细胞发生分化,还要在培养液中加入白血病抑制因子(LIF),胎牛血清的质量及浓度也是影响ES细胞生长状况的重要因素,为此本实验采用GIBCO公司生产的胎牛血清,所加浓度为15%。形成的ES细胞样集落,继续培养后观察集落的生长状态,并通过碱性磷酸酶染色,在体外形成拟胚体,移植入裸鼠皮下4周后取材,组织学观察进行鉴定。结果:ES细胞有其典型的形态学特征:集落呈鸟巢状,边缘清楚,表面平滑,结构致密,隆起生长,细胞之间界限不清楚;单个细胞体积小、核大;对ES细胞碱性磷酸酶进行检测,在AKP底物作用下,未分化的ES细胞显微镜下为棕褐色,分化的不着色。悬滴法培养ES细胞两天后生成拟胚体,它是胚胎干细胞在体外一定条件下自发形成的类似早期胚胎的球体结构,早期发育的简单拟胚体包含了外层的原始内胚层和内层的原始外胚层,这种结构与胚胎发育过程中的卵圆柱期结构十分相似。体内研究表明,将小鼠胚胎干细胞注入裸鼠皮下4周后,可形成含有三胚层组织的畸胎瘤。结论:胚胎干细胞具有高度的复制能力及多向分化的潜能。 2.成釉细胞条件培养液诱导小鼠ES细胞分化的研究目的:探讨成釉细胞构建的微环境对于小鼠胚胎干细胞的诱导能力。方法:本实验胚胎干细胞采用两种途径分化:生成拟胚体(EB)与不生成拟胚体直接诱导分化的途径,利用发育早期成釉细胞分泌的信号分子在体外模拟成牙微环境,观察对小鼠胚胎干细胞增殖分化的影响。我们用成釉细胞的条件培养液诱导胚胎干细胞,并选择两个时间点,7天和14天,从形态学观察分化后细胞的形态特征,并从基因水平和蛋白水平检测诱导后细胞的表型变化。随后我们将两个诱导组的细胞植入裸鼠皮下4周观察体内结果,并将出生1天的C57小鼠的成釉器同样植入裸鼠皮下作为阳性对照。结果:体外利用成釉细胞无血清条件培养液诱导小鼠胚胎干细胞,形态学观察诱导后可促进其增殖和分化,诱导后的部分细胞形态与成釉细胞形态相似。随后的基因或蛋白检测发现诱导后的细胞具有牙源性上皮细胞的表型。其中经过EB诱导分化途径比直接诱导分化途径的效率要高一些。RT-PCR结果表明诱导后的胚胎干细胞表达牙上皮相关蛋白CK14、AMBN及AMGN,免疫荧光结果及Western Blot结果与RT-PCR结果一致。体内实验更验证了体外的结果,将诱导后的小鼠胚胎干细胞经纤维蛋白胶包裹移植于裸鼠皮下,培养4周后观察经过EB诱导分化途径的细胞在体内可形成一些上皮样角化组织,进一步免疫组织化学染色显示角化组织可表达CK14、AMBN、AMGN,具有类似成釉器样组织植入裸鼠皮下生成组织的特性。然而不经过EB诱导分化途径的细胞在体内生成了大量的结缔组织,未见典型的上皮样组织结构。当通过拟胚体途径经ASF-CM条件培养液诱导14天的胚胎干细胞与小鼠牙胚细胞混合植入裸鼠皮下后,结果显示,诱导后的胚胎干细胞在牙胚细胞的诱导下,参与了矿化组织的形成,因为经CFSE标记带绿色荧光的胚胎干细胞与矿化组织部分重合。未诱导的胚胎干细胞与牙胚细胞混合后,体内结果显示移植物仍然生成畸胎瘤。结论:以上结果提示,成釉细胞无血清条件培养液所提供的成牙微环境可在体外和体内促进小鼠胚胎干细胞的牙向分化,通过拟胚体途径的分化效率更高一些,且通过拟胚体途径诱导后的胚胎干细胞在体内能够生成牙源性上皮样组织,提示我们胚胎干细胞向牙源性上皮方向转化的可能性。 3.牙胚细胞条件培养液诱导小鼠ES细胞分化的研究目的:探讨牙胚细胞构建的微环境对于小鼠胚胎干细胞的诱导能力。方法:本实验胚胎干细胞采用两种途径分化:生成拟胚体(EB)与不生成拟胚体直接诱导分化的途径,利用发育早期牙胚细胞分泌的信号分子在体外模拟成牙微环境,观察对小鼠胚胎干细胞增殖分化的影响。本实验主要从形态学、基因及蛋白水平进行检测,并将诱导后的细胞移植入体内进行观察。结果:体外利用牙胚细胞条件培养液诱导培养小鼠胚胎干细胞,通过两种分化途径,并未实现向牙源性上皮细胞方向的表型转化。形态学观察经过牙胚条件培养液诱导后,两种途径诱导的细胞增殖能力缓慢,且诱导14天后细胞出现凋亡。基因表达仅表达DMP1和CK14,未表达牙齿上皮样相关基因。体内移植物为疏松的结缔组织,少量移植物体内生成畸胎瘤。未见成釉细胞及牙齿相关组织形成。结论:以上结果提示,与成釉细胞条件培养液相比,牙胚细胞条件培养液所提供的成牙微环境并不能促进小鼠胚胎干细胞的牙向分化。
[Abstract]:The process of tooth development depends on the mutual induction between the epithelial tissue that is located at the location of the tooth and the mesenchyme at the lower part of the tooth. Therefore, the biological regeneration of the teeth requires at least two kinds of cells: the odontogenic epithelial cells needed for the formation of enamel and the odontogenic mesenchymal cells forming the dental pulp complex. So far, there are many alternative studies on odontogenic mesenchymal cells, such as adult dental pulp stem cells, bone marrow stromal cells, dermal dermal cells and hair follicle cells, which have been proved to have the ability to convert to odontogenic mesenchymal cells. However, the search for the successful substitution of appropriate cells to odontogenic epithelial cells is more successful. Although some scholars believe that the cells derived from adult bone marrow can convert to ameloblastoma, the ability to convert two cells to ameloblastoma like cells and odontogenic mesenchymal cells restricts its application. This study must be based on the use of an embryonic or 1 day autologous oral and palatine epithelium, which also restricts its application. This limitation encourages us to find a new cell to differentiate into the odontogenic epithelium. The ability of embryonic stem cells to be self renewing and pluripotent has received many studies in recent years. Some studies have shown that embryonic stem cells can differentiate into skin epidermal cells, lung epithelial cells and thymic epithelial cells, but it is not yet reported whether embryonic stem cells can convert to odontogenic epithelium. Therefore, the answer to this question is undoubtedly to solve the epithelic seeds in dental regeneration. The bottleneck problem of cell origin is of great significance.
1. culture and identification of embryonic stem cells
Objective: to develop and identify the R1 and CGR8 lines of embryonic stem cells. Methods: embryonic stem cells are very susceptible to external factors and are very demanding for growth conditions. The normal embryonic stem cell culture contains high sugar MEM medium, fetal bovine serum, L- glutamine, 2- mercapto ethanol and non essential amino acids, in order to prevent embryos from embryos. The stem cells are differentiated and the leukemia inhibitory factor (LIF) is added to the culture medium. The quality and concentration of fetal bovine serum are also the important factors affecting the growth of ES cells. Therefore, this experiment adopts the fetal bovine serum produced by GIBCO company, and the concentration is ES fine cell like colony formed by 15%., and the growth state of the colony is observed after continuing culture. With alkaline phosphatase staining, the embryoid body was formed in vitro, and transplanted into the subcutaneous tissue of nude mice for 4 weeks. The results showed that ES cells had its typical morphological characteristics: the colony was nests, the edges were clear, the surface was smooth, the structure was dense, the growth of the cells was not clear, the single cell was small, and the nucleus was large; and ES Cell alkaline phosphatase was detected, under the action of AKP substrate, the undifferentiated ES cell was brown in the microscope, and the differentiation was not coloured. The suspension method was used to produce the ES cells for two days to produce the embryoid body. It was a ball structure similar to the early embryo, which was formed spontaneously under certain conditions in vitro. The original ectoderm of the outer layer of the primordial endoderm and the inner layer of the inner layer, which is very similar to the egg cylindrical structure during the embryonic development, has shown that the mouse embryonic stem cells can form a teratoma containing three germ layers after 4 weeks of subcutaneous injection in nude mice. Conclusion: embryonic stem cells have high replication ability and multidirectional fraction. The potential of transformation.
Differentiation of mouse ES cells induced by 2. ameloblastoma conditioned medium
Objective: To investigate the induction ability of the microenvironment constructed by ameloblastoma to mouse embryonic stem cells. Methods: the experimental embryonic stem cells were divided into two pathways: the generation of EB and the direct induction of differentiation by the non embryoid body, and the observation of the microenvironment by using the early ameloblastoma signal molecules to simulate the microenvironment in vitro. The effect on the proliferation and differentiation of mouse embryonic stem cells. We use the conditioned medium of ameloblastoma to induce embryonic stem cells, and select two time points, 7 days and 14 days, observe the morphological characteristics of the cells after differentiation, and detect the phenotypic changes of the cells after the induction of the gene level and protein level. Then we will take two induction groups. The cells were implanted in the nude mice for 4 weeks to observe the results of the body, and the glaze apparatus of the C57 mice, which was born 1 days, was implanted subcutaneously as a positive control. The morphology of ameloblastoma was similar. Subsequent gene or protein detection found that the induced cells had phenotypes of odontogenic epithelial cells. The EB induced differentiation pathway was more efficient than the direct induction of differentiation pathway..RT-PCR results showed that the induced embryonic stem cells expressed CK14, AMBN and AMGN, and immunofluorescence. The results and the results of Western Blot were in accordance with the results of RT-PCR. In vivo, the results were verified in vitro, and the induced mouse embryonic stem cells were transplanted subcutaneously in nude mice with fibrin glue. After 4 weeks of culture, the cells with EB induced differentiation could form some epitheliated keratinized tissues in the body and further immuno histochemistry The staining showed that the keratinized tissue could express CK14, AMBN, AMGN, which had the characteristics of implanting subcutaneous tissue in nude mice like the enamel like tissue. However, a large number of connective tissues were produced in the body without EB induced differentiation, and no typical epithelioid structure was found. 14 days after the passage of the embryoid pathway through the ASF-CM conditioned medium The results showed that the induced embryonic stem cells were involved in the formation of mineralized tissues under the induction of tooth germ cells, because the CFSE labeled embryonic stem cells with green fluorescence coincided with the mineralized tissue, and the uninduced embryonic stem cells were mixed with the tooth germ cells. The results show that the teratoma is still generated by the graft. Conclusion: the above results suggest that the dental microenvironment provided by the serum-free culture medium of ameloblastoma can promote the tooth differentiation of mouse embryonic stem cells in vitro and in vivo, and the differentiation efficiency through the embryoid pathway is higher, and the embryo fines are induced by the embryoid pathway. Cells can generate odontogenic epithelioid tissue in vivo, suggesting the possibility of embryonic stem cells transferring to the odontogenic epithelium.
Differentiation of mouse ES cells induced by 3. dental germ cell conditioned medium
Objective: To explore the induction ability of the microenvironment constructed by tooth germ cells for mouse embryonic stem cells. Methods: the experimental embryonic stem cells were divided into two ways: the generation of the embryo (EB) and the direct induction of differentiation from the non embryoid body, and using the signal molecules of the early developmental tooth germ cells to simulate the tooth microenvironment in vitro, and observe the microenvironment in vitro. The effect of the experiment on the proliferation and differentiation of mouse embryonic stem cells. This experiment was mainly detected from morphology, gene and protein level, and the induced cells were transplanted into the body for observation. Results: the mouse embryonic stem cells were induced and cultured in vitro by the condition culture solution of tooth germ cell, and the odontogenic epithelium was not finely realized through two differentiation pathways. Phenotypic transformation in the direction of the cell. Morphological observation was induced by the conditioned medium of tooth germ. The cell proliferation ability induced by two pathways was slow and apoptosis was induced in 14 days. Gene expression only expressed DMP1 and CK14 and did not express the epithelioid related genes in the teeth. The body was loose connective tissue and a few transplants were formed to produce malformation. There is no formation of ameloblastoma and dental related tissue. Conclusion: the above results suggest that the dental microenvironment provided by dental germ cell conditioned medium can not promote the tooth differentiation of mouse embryonic stem cells compared with the conditioned medium of ameloblastoma.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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