請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62810完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳敏慧 | |
| dc.contributor.author | Yun-Yuan Tai | en |
| dc.contributor.author | 戴允元 | zh_TW |
| dc.date.accessioned | 2021-06-16T16:11:07Z | - |
| dc.date.available | 2018-03-04 | |
| dc.date.copyright | 2013-03-04 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-02-19 | |
| dc.identifier.citation | References
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62810 | - |
| dc.description.abstract | [研究目的]
上皮層增厚並套疊進入間葉層(Epithelial invagination)是外胚層器官發育機轉(Ectodermal Organogenesis)的開始。雖然有許多生長因子被發現可調控外胚層器官，但對於它們即時性(real-time)的作用卻無法提供深入了解。細胞與基質電阻抗偵測系統(Electric cell-substrate impedance sensing, ECIS)是一種不具侵入性、即時偵測細胞或器官變化的系統，可利用細胞在微電極上介面電阻的改變量以即時偵測器官或細胞的厚度改變。過去常用以偵測癌症細胞入侵組織器官的變化。由於上皮細胞向間葉層套疊過程近似入侵，是以極適合用以偵測該現象。本研究目的即建立重組器官電阻抗即時偵測模組(bioengineered organ-ECIS model)，藉以即時偵測第九號纖維母細胞成長因子(FGF-9) 加速外胚層器官上皮增厚向間葉套疊(epithelial invagination)進程以誘發外胚層器官發育的機轉。 [研究方法] 吾人利用已知的生物工程外胚層器官重組技術(bioengineered ectodermal organ)，將第14天大的老鼠胚胎內第一大臼齒牙胚取出並切開上皮與間葉，利用細胞重組技術再生外胚層器官，將其培養在細胞與基質電阻抗偵測系統(ECIS Z8)微電極上，以即時偵測第九號纖維母細胞成長因子(FGF-9) 在外胚層器官發育過程所扮演的角色。 [研究成果] 實驗證明第九號纖維母細胞成長因子(FGF-9)不僅可在三天內加速再生牙胚上皮增厚向間葉套疊(epithelial invagination)的過程，也同時刺激再生牙胚分泌更多齒釉蛋白（amelogenin）與成釉蛋白（ameloblastin）。由此可見第九號纖維母細胞成長因子(FGF-9)在誘發外胚層器官發育過程中的主導性。亦證明吾人所建構之重組器官電阻抗即時偵測模組(bioengineered organ-ECIS model)可有效達成即時偵測特定成長因子(如FGF-9)對外胚層器官發育過程所造成的影響，進而有利未來深入研究相關器官發育機轉。 [成果討論與總結] 本研究是首次以即時偵測系統證實第九號纖維母細胞成長因子(FGF-9)可藉由促進間葉細胞間交互整合、刺激上皮細胞增生變厚與分化為成齒釉細胞層(ameloblast layer)以加速牙胚上皮增厚向間葉套疊(epithelial invagination)的過程，並進而誘發外胚層器官發育機轉(Ectodermal Organogenesis)。 由此可知纖維母細胞成長因子(FGFs)族群在外胚層器官發育過程中佔有舉足輕重之地位，並可進而用以加速未來外胚層器官再生進程。基於以上研究成果，吾人據此提出纖維母細胞成長因子(FGFs)與骨構型蛋白(BMPs)在外胚層器官發育過程中同樣佔有主導地位。並依據本次研究成果與過去先輩之研究發現，倡議可調控外胚層器官型態之｀纖維母細胞成長因子與骨構型蛋白拮抗調控系統＇(FGF-BMP Balancing System)。 | zh_TW |
| dc.description.abstract | [Introduction]
Epithelial invagination is important for initiation of ectodermal organogenesis. Although many factors regulate ectodermal organogenesis, there is not any report about their functions in real-time study. Electric cell-substrate impedance sensing (ECIS), a non-invasive, real-time surveillance system, had been used to detect changes in organ cell layer thickness through quantitative monitoring of the impedance of a cell-to-microelectrode interface over time. It was shown to be a good method for identifying significant real-time changes of cells. The purpose of this study is to establish a combined bioengineered organ-ECIS model for investigating the real time effects of fibroblast growth factor-9 (FGF-9) on epithelial invagination in bioengineered ectodermal organs. [Materials and Methods] We dissected epithelial and mesenchymal cells from stage E14.5 murine molar tooth germs and cultured the regenerated tooth germ with bioengineered organ germ method in the ECIS Z8. We identified the real-time effects of FGF-9 on epithelial-mesenchymal interactions using this combined bioengineered organ-ECIS model. Then we checked the Ameloblastin and Amelogenin expression with 7900HT real-time polymerase chain reaction in different time spots. [Results] Measurement of bioengineered ectodermal organ thickness showed that FGF-9 accelerates epithelial invagination in reaggregated mesenchymal cell layer within 3 days. Gene expression analysis revealed that FGF-9 stimulates and sustains early Ameloblastin and Amelogenin expression during odontogenesis. [Discussion and Conclusions] This is the first real-time study to show that, FGF-9 plays an important role in epithelial invagination and initiates ectodermal organogenesis—by facilitating cross-talk and reunion between mesenchymal cells and stimulating epithelial cell layer differentiation, creating an ameloblast layer. Based on these findings, we suggest FGF-9 can be applied for further study in ectodermal organ regeneration, and we also proposed that the ‘FGF-BMP balancing system’ is important for manipulating the morphogenesis of ectodermal organs. The combined bioengineered organ-ECIS model is a promising non-invasive real-time method for ectodermal organ engineering and regeneration research. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T16:11:07Z (GMT). No. of bitstreams: 1 ntu-102-Q96422003-1.pdf: 6168931 bytes, checksum: 5ec8868f0d2a6609520513d6d906767b (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 目 錄
口試委員會審定書……………………………………………………………… i 誌謝………………………………………………………………………………. ii 中文摘要………………………………………………………………………… iii 英文摘要…………………………………………………………………………. v Chapter 1. Introduction: Investigating Ectodermal Organogenesis from Tooth Germ Model........................................................................................................................ 1 1.1. Preface: Curiosities about Ectodermal Organogenesis ....................………………. 1 1.2. Significant Research Works about Ectodermal Organogenesis................................... 2 1.2.1. Morphogenesis of Ectodermal Organs…………………………………………… 2 1.2.2. From “Non-dental” to “Dental”!!!......................................................................... 5 1.2.2.1. BMPs—Inducing Diastema Teeth………………………………………………. 6 1.2.2.2. Eda-Edar(TNFs) —Inducing Diastema Teeth…………………………….. 6 1.2.2.3. Shh–BMP ‘Signalling Module’?.................................................................. 8 1.2.2.4. BMPs--Inducing Supernumerary Anterior Teeth…………………………. 9 1.2.2.5. Wnts—Inducing Supernumerary Anterior Teeth………………………….. 10 1.2.3. How to differentiate between “Non-dental” and “Dental” ?............................ 12 1.2.3.1. Wnt-7b Inhibits Shh in the Diastema (Non-dental) Area…………………. 12 1.2.3.2. Wnts—Inducing Diastema Teeth………………………………………….. 13 1.2.3.3. “Fine-Tuning Regulation System” for Shh Expression?!........................... 15 1.2.4. Role of FGFs For Ectodermal Organogenesis.................................................. 17 1.3. Summaries and Mysteries: Possible Fine-Tuning Regulatory System for Ectodermal Organogenesis......................................................................................... 19 1.4. Target of This Study: FGF-9........................................................................................ 21 Chapter 2. Materials and Methods: The Bioengineered Organ-ECIS Model.................... 22 2.1. Why We Need A New Model?....................................................................................... 22 2.2. Introduction of Electric Cell-substrate Impedance Sensing (ECIS)…………......... 23 2.3. Animals........................................................................................................................... 25 2.4. First Molar Tooth Germ Dissection and Dissociation into Single Cells through the Non-serum Protocol………………………………………….... 26 2.5. Determine the Optimum Concentration of FGF-9 from MTT Assay…………...... 29 2.6. Bioengineered Organ Culture via Electric Cell-Substrate Impedance Sensing (Bioengineered Organ-ECIS Model)................................................. 30 2.7. 7900HT Real-time Polymerase Chain Reaction Gene Expression Assay and Statistical Analysis………………………………………………………. 30 Chapter 3. Results: FGF-9 Accelerates Epithelial Invagination for Ectodermal Organogenesis…………………………………………………………………… 32 3.1. FGF-9 Promotes Tooth Germ Development…..…….…………………………….... 32 3.2. ECIS impedance change reveals FGF-9 stimulates accelerated epithelial invagination……………………………………………………………………... 33 3.3. FGF-9 accelerates and sustains Ameloblastin and Amelogenin expressions.............. 35 Chapter 4. Discussion and Conclusion: FGF-BMP Balancing System................................. 38 4.1. Discussion……………………………………………………………………………… 38 4.2. Conclusion……………………………………………………………………………... 41 Chapter 5. Future Aspects: Regenerating Ectodermal Organs........................................... 43 5.1. Discover sources of ectodermal epithelial cells for new organ(s) regeneration....... 43 5.2. Discover controlling regulatory systems for ectodermal organogenesis.................. 43 5.3. Discover new scaffold(s) for regenerating ectodermal organs.................................. 45 References…………………………………………………………………………………….. 47 Appendix 1: Additional Files from Preliminary Experiments…………………………….. 57 Additional file 1: Preliminary experiment: FGF-9 upregulates Ameloblastin and Amelogenin in cultured mesenchymal cells………………………… 57 Additional file 2: Haematoxylin and eosin staining of ectodermal organ culture.......... 59 Additional file 3: Haematoxylin and eosin staining of ectodermal organ culture.......... 60 Appendix 2: Supplements of Protocols…………………………………………………….... 61 A1. Get E14.5 mouse embryos for study............................................................................ 61 A1.1. Prepare “Zoleteil 50” ........................................................................................ 61 A1.2. Get E14.5embryo from pregnant female mice................................................ 61 A1.3. Special notes for tooth germ dissection and separation of epithelium and mesenchyme.............................................................................................. 61 A2. Prepare different FGF-9 concentrations.................................................................... 62 A3. Tips for ECIS assay...................................................................................................... 62 A3.1. Change medium during the assay.................................................................... 62 A4. 7900HT real-time PCR................................................................................................. 62 A4.1. RNA Extraction with PurelinkTM RNA Mini Kit + TRIzol Reagent............ 63 A4.2. Reverse Transcription from RNA to c-DNA with High Capacity RNA-to-cDNA Kit........................................................................................... 64 A4.3. Real-Time PCR with TaqMan ® Gene Expression Assay and Master Mix in 7900 HT........................................................................................................ 65 A4.4. Materials Prepared for RNA Extraction, Reverse Transcription, and 7900HT Real-Time PCR................................................................................. 66 圖目錄 Summary Graph. A. Possible regulating mechanism during the tooth germ development and ectodermal organogenesis................................................................ 5, 11, 19 Summary Graph. B. Can we enlarge tooth crown size or regenerate extra teeth through p21 overexpression and suppression of Ectodin?........................................7, 11, 19 Summary Graph. C. The dental and nondental border might be determined by the antagonistic expression of Wnt and Shh..................................................................... 16, 20 Fig. 1. Tooth germ dissection and dissociation…………..……………………………….….. 23 Fig. 2. Mechanism of ECIS assay………………..………..……………………………….….. 24 Fig. 3. The combined bioengineered organ-ECIS model for analysis of epithelial invagination 25 Fig. 4. Check the pregnancy of female mouse………………..………..…………………..….. 26 Fig. 5. The stereomicroscope and E14.5 embryos taken for further microsurgery…………... 26 Fig. 6. E14.5 embryo and the dissected first molar tooth germ………………………………. 27 Fig. 7. H & E stain of E14.5 first molar tooth germ………………………………………….. 27 Fig. 8. Multiphoton picture of E14.5 first molar tooth germ…………………………………. 28 Fig. 9. Three steps of dissecting the first molar tooth germ to separate the epithelium and mesenchyme………………………………………………………………………….. 28 Fig. 10. Protocol of Non-Serum Dissociation Method………………………………………. 29 Fig. 11. MTT assay…………………………………………………………………………... 33 Fig. 12. ECIS Assay………………………………………………………………………….. 35 Fig. 13. Real-time PCR measurement of Ameloblastin and Amelogenin gene expression….. 37 Fig. 14. FGF-BMP Balancing System regulates morphogenesis of ectodermal organs…... 42 Fig. 15. Regenerative Medicine can be started from the Bioengineered Organ Model.......... 45 Additional file 1. Preliminary experiment: Fibroblast growth factor-9 (FGF-9) upregulates Ameloblastin and Amelogenin in cultured mesenchymal cells.................... 57 Additional file 2. Haematoxylin and eosin staining of ectodermal organ culture. Day-1....... 59 Additional file 3. Haematoxyliin and eosin staining of ectodermal organ culture. Day-10.... 60 | |
| dc.language.iso | en | |
| dc.subject | 外胚層器官發育機轉 | zh_TW |
| dc.subject | 纖維母細胞成長因子與骨構型蛋白拮抗調控系統 | zh_TW |
| dc.subject | 第九號纖維母細胞成長因子 | zh_TW |
| dc.subject | 上皮層向間葉層套疊 | zh_TW |
| dc.subject | 重組器官電阻抗即時偵測模組 | zh_TW |
| dc.subject | FGF-9 | en |
| dc.subject | Epithelial invagination | en |
| dc.subject | Ectodermal organogenesis | en |
| dc.subject | Combined bioengineered organ-ECIS model | en |
| dc.subject | FGF-BMP balancing system | en |
| dc.title | 第九號纖維母細胞成長因子加速上皮套疊入間葉層以誘發外胚層器官發育機轉 | zh_TW |
| dc.title | FGF-9 Accelerates Epithelial Invagination For Ectodermal Organogenesis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 張富雄,林泰元,劉宏輝,王志龍,賴泰廷 | |
| dc.subject.keyword | 第九號纖維母細胞成長因子,上皮層向間葉層套疊,外胚層器官發育機轉,重組器官電阻抗即時偵測模組,纖維母細胞成長因子與骨構型蛋白拮抗調控系統, | zh_TW |
| dc.subject.keyword | FGF-9,Epithelial invagination,Ectodermal organogenesis,Combined bioengineered organ-ECIS model,FGF-BMP balancing system, | en |
| dc.relation.page | 66 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-02-19 | |
| dc.contributor.author-college | 牙醫專業學院 | zh_TW |
| dc.contributor.author-dept | 臨床牙醫學研究所 | zh_TW |
| 顯示於系所單位： | 臨床牙醫學研究所 | |
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