纖維母細胞生長因子-9培養胎鼠牙胚幹細胞之研究

牙齒在攝取營養、外觀及與人溝通方面，都是不可或缺的。齲齒、牙髓炎、牙周疾病及外傷等因素，將導致牙齒缺失，進而影響正常咀嚼、發音等生理功能，並造成外型上的困擾。若是能發展出生物性的替代牙齒便能取代缺損的牙齒，將是臨床的一大突破。經過幾年來的研究，人們對於牙齒的再生有了更深一層的了解，很多藉由動物的牙齒胚胎來了解牙胚細胞在經過胚層分離、培養最後重新組合成一顆牙齒類似物，其中所運用到的特定的生長激素、某些基因的抑制與表現…等等。主要的概念就是希望經由體外的牙胚細胞重組、調控，最終目的就是希望能夠再生一顆有價值的生物工程牙胚。根據最近發表的研究結果，有人找出利用基因轉殖技術，將四種主要的基因插入到fibroblast中，形成所謂的IPS細胞(induced pluripotent stem cells)，在實驗組中竟然可以找到許多跟胚胎幹細胞相似的特性，這種利用藉由插入基因而增加細胞特性的方法，讓器官再生醫學又找到一條可行的道路。本篇實驗主要在研究牙齒再生及特定生長因子對於牙胚細胞生長過程的影響，實驗從懷孕母鼠體內胚胎14天之胎鼠下顎經過立體顯微鏡及外科手術取得臼齒的牙胚細胞，經過酵素作用把完整的牙胚切割成上皮細胞及間葉細胞，一方面建立MTT找出對牙胚細胞有適當影響的Fgf-9的濃度。另外將分離的牙胚細胞培養在Transwell® 中，利用相同的五個時間點及實驗組對照組來檢驗期間的特定基因RNA變化量，來推論特定濃度的Fgf-9對間葉細胞在分化以及增生上的影響，最後利用H&E染色切片來說明經過器官培養十天的對照組與實驗組他們外觀上的差異。本研究結果發現 : (1)纖維母細胞生長因子-9在濃度為25 ng/ml的情況下，對於間葉細胞的生長情況為最佳也最有效率；(2)利用同為牙胚幹細胞的上皮細胞與間葉細胞進行co-culture培養，其中實驗組加入濃度為25 ng/ml纖維母細胞生長因子-9(Fgf-9)個別培養一天、四天、七天、十四天與十六天後，從mRNA的表現來看，的確有增加、增快間葉細胞產生日後鈣化的重要蛋白質之基因表現；(3)從器官培養的實驗中可以觀察到有加入纖維母細胞生長因子-9的組別比control組更快進入分泌前期，從切片中可以看到類似牙釉質的分泌物。因此可以推論，在本實驗的co-culture中加入生長因子-9，會幫助間葉細胞加速分化。綜合本研究結果可知：纖維母細胞生長因子-9可以促使牙胚間葉細胞表現成牙本質表型特徵，至於纖維母細胞生長因子-9是否可以有效地促使並且加速重組牙胚的再生則需要更完整的研究數據。

關鍵字

纖維母細胞生長因子-9 ；牙胚幹細胞；間葉細胞；成牙本質化；器官培養

並列摘要

The human dentition is indispensable for nutrition, communication and physiology. Caries, pulpitis, apical periodontitis and trauma may lead to loss of tooth, causing the problem of pronunciation, mastication, and appearance difficulities. Abiological tooth substitute that could replace lost teeth would provide a vital alternative to currently avalible clinical treatment. For years research, people have obtained much more knowledge about this field. By animal model of embryonic tooth germ, we can understand how tooth germ cells separation of embryo layer, culture and finally recombination into tooth-like organ. In this experiment, we use some specific growth factors, some genetic inhibition and genetic expression., etc. Main conception is hoping through recombination and regulation of tooth germ cells in vitro. Our final subject is to regenerate a valuable bio-engineering tooth. According to the recent research, they inerted four significant genes into fibroblast with genetic transfection technology which forms “IPS”cells (induced pluripotent stem cells). In experiment group, they can find many properties similar to embroyo-stem cells. By inserting genes into fibroblast, we could find an alternative way in organic-regeneration-medication. This study focues on observation of tooth regeneration and the effect of specific growth factor treated on tooth germ cells. We obtained tooth germ stem cells form embryonic day 14th mouse low jaw molars through stereo-microscope. Then we minced and separated impact tooth germ into epithelium and mesenchyme stem cells by enzyme treatment. In addition, we find out the proper concentration of fibroblast growth factor-9 by MTT assay. We cultured separated tooth germ cells in TRANSWELL system and set up five different points and two groups, one is control group, and the other is experiment group, to observe the difference in RNA expression, at last we can discuss the effect on mesenchyme cells treated with Fgf-9. To observe the histology we use Hematoxylin and eosin (H&E) paraffin section of organ culture for ten days in Fgf-9 medium to compare with control group. Our results demonstrated that: (1) According to MTT assay, mesenchyme cells perform highly effective under circumstances with 25 ng/ml Fgf-9 in medium. (2)The expression of mesenchyme calcification-gene mRNA obviously increased from first day to sixteenth day co-cultured with 25 ng/ml Fgf-9 medium.(3)We can clearly distinguish the difference between control group and experiment group which proceed to secrete enamel-like matter. These results suggest that: dental mesenchyme cells with odontogenic cells phenotype stimulated by fibroblast growth factor-9 could promote odontogenic gene expression. Whether or not fibroblast growth factor-9 can effectively promote and accelerate the regeneration of recombination-tooth needs more detailed study. Keywords: fibroblast growth factor-9 , dental stem cells , mesenchyme cells , odontogenic , organ culture.

並列關鍵字

fibroblast growth factor-9 ； dental stem cells ； mesenchyme cells ； odontogenic ； organ culture

參考文獻

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纖維母細胞生長因子-9培養胎鼠牙胚幹細胞之研究

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