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標題: | 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 FGF-9 Accelerates Epithelial Invagination For Ectodermal Organogenesis |
作者: | Yun-Yuan Tai 戴允元 |
指導教授: | 陳敏慧 |
關鍵字: | 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, FGF-9,Epithelial invagination,Ectodermal organogenesis,Combined bioengineered organ-ECIS model,FGF-BMP balancing system, |
出版年 : | 2013 |
學位: | 博士 |
摘要: | [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)。 [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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62810 |
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顯示於系所單位: | 臨床牙醫學研究所 |
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