【作者】 闫征斌；

【导师】 田卫东；

【作者基本信息】 四川大学 ， 口腔临床医学， 2005， 博士

【摘要】 最新的发育生物学研究证实,在颅神经嵴细胞分化形成牙颌骨器官的过程中,先后经历了颅神经嵴→第一鳃弓外胚间充质→牙颌骨外胚间充质两次关键性级联分化。本研究旨在建立Balb/c胎小鼠第一鳃弓外胚间充质细胞体外培养模型,在此基础上,进行多向诱导分化研究,鉴定其干细胞生物学特性。同时,构建第一鳃弓外胚间充质细胞牙向分化双向抑制消减文库,初步筛选、验证、分析差异表达基因,为全面揭示牙发生、发育的分子调控机制提供理论依据。 首先,精确解剖分离孕9.5天(E9.5)Balb/c胎鼠第一鳃弓,采用组织块法和改良酶消化法对其进行原代培养,利用反复贴壁和多次差异消化法对其进行纯化,通过细胞形态观察、细胞生长曲线描记、计算细胞倍增时间和特异性标记物检测对其细胞生物学特性进行研究。其次,利用端粒酶原位杂交荧光探针和Brdu摄取实验,检测第一鳃弓外胚间充质细胞的增殖和自我更新能力;取第3代细胞,更换不同的条件培养液,观察其在成脂、成内皮和矿化诱导液的刺激下,细胞形态学变化,并检测鉴定相应的特异性细胞表型标记,对细胞的可塑性进行研究。然后,在相同的培养条件下,收取第3代第一鳃弓外胚间充质细胞和E16.5下颌第一磨牙牙乳头细胞,采用抑制消减杂交技术,构建双向抑制消减文库,并对细胞的总RNA质量、双链cDNA酶切效果、接头连接率、抑制消减效率、差异基因的富集程度和载体连接效率进行全面质控分析。最后,在双向抑制消减文库中随机挑更多还原

【Abstract】 The latest research of developmental biology indicated, Cranial Neural Crest Cells (CNCCs) contributed significantly to the formation of craniofacial structures during embryonic development. In this process, CNCCs underwent two key cascade differentiation, CNCC — the first branchial arch ectomesenchyme (FBAEM) — oral and maxillofacialectomesenchyme(OMFEM).This study was aimed to isolate and culture the mesenchyme cells from the first branchial arch of the fetal mice, and to confirm the molecular and cellular characterization of ectomesenchymal cells during multilineage differentiation in vitro. At the same time, the double-direction suppression subtractive hybridization (SSH) cDNA libraries of odontogenic differentiation of FBAEMCs were constructed and primary detection and analysis of differerntially expressed genes were carry out. It will provide the basis of theoretics for regulation mechanism of tooth development furthely.Firstly, the first branchial arch primordia from E9.5 embryos were dissected under microscope. Explanted culture and improved enzyme digestion were all used to primary culture. The growth curve, cells doubling time, cellsproliferation and morphology were studied, immunocytochemistry assay was used to identify the source and the state of FBAEMCs. Secondly, the activity of proliferation and self-renewal of FBAEMCs were detected by nuclear labeled with BrdU and fluorescence in situ hybridization for telomerase. Monolayer cultures of ectomesenchymal cells were passaged 3 times and then transferred to adipogenic,endothelial and osteogenic media by using a combination of previously reported protocols for other species. The level of differentiation was evaluated by histological examination and by analyzing the expression of tissue-specific genes by reverse transcription/polymerase chain reaction technique. Thirdly, the double-direction SSH cDNA library of the 3rd passage of FBAEMCs and the dental papillae cells isolated from 1st lower molar tooth germs were built up. Analysis of total RNA qualities, cDNA Taq I digestion, adaptors ligation efficiency, subtraction efficiency and PCR products were all studied. Finally, 23 clones were randomly selected to sequence and analyzed for homology in the GeneBank. Quantitative analysis of differentially expressed genes were carried out by real-time PCR in E9.5, E12.5, E14.5 and E16.5.The nasofrontal process, the first branchial arch and the second branchial arch in E9.5 were very evident and easy to dissected. There were more epithelial-like cells in the explant primary culture, the cells were grown to confluency for 7-10 days. Improved enzyme digestion of primary culture has less epithelial-like cells, the cells were grown to confluency for 2-3 days. The growth curve, cell doubling time and morphology of two methods after passage were similar. Anti-CD57/HNK-1 and anti-Vimentin were all positive. The high activity of proliferation and self-renewal of FBAEMCs were observed by nuclear labeled with BrdU and fluorescence in situ hybridization for telomerase. After beingcultured in an adipogenesis-inducing medium, the ectomesenchymal cells responded by the accumulation of lipid vacuoles and the gene expression of LPL. Following osteoinduction, the isolated fibroblast-like cells transformed into cuboidal cells, and formed mineralized nodules. In addition, osteogenesis was followed by collagen type I immunocytochemica staining and ALP staining. Epithegenesis was determined by vWF immunocytochemical staining. FBAEMCs qua tester, there were 184 white clones in the forward direction subtractive libraries, FBAEMCs qua driver, there were 132 white clones in the reverse direction subtractive libraries. The double-direction libraries have high quality by results of SSH analysis. After sequencing, the repeated sequences were removed, 15 fragments were analyzed for homology, the results showed 11 fragments were part of the known genes of mouse, 4 fragments were DNA sequences and likely to unknown new genes. The results of real-time PCR of seven differently expressed genes were the same as the results of SSH. Change of clone 1 -3 ralative copies was very prominent, thereby ulteriorly demonstrate it was a new gene.In conclusion, culture model of ectomesenchyme cells isolated from the first branchial arch was successfully established in vitro. Sufficient and high purity ectomesenchyme cells could be harvested in short time by improved enzyme digestion primary culture. FBAEMCs had high activity of proliferation and self-renewal and were capable of in vitro extensive multiplication and multilineage differentiation, making them a relevant and invaluable model in the field of stem cell research and the orofacial development. Then high quality subtractive cDNA libraries of odontogenic differentiation of FBAEMCs were triumphantly constructed by SSH for the first time. The functions ofdifferentially expressed genes were primary discussed by bioinformatics. The results of SSH libraries were validated once again by real-time PCR on the histology level. It will provide the basis of theoretics for cloning the new gene.更多还原