論文名稱:

應用活化基因基質技術於顱頂骨再生之基礎研究

 

Application of Gene-activated Matrix in Craniofacial Bone regeneration

研究生:

張筱河  Hsiao-he Chang

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

陳恆理  Hen-Li Chen

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

39217009

          學年度:

93

          語文別:

中文

          出版年:

94

關鍵字:

活化基因基質  Gene-activated matrix

 

膠原蛋白  collagen

 

副甲狀腺素  parathyroid hormone

全文說明:

(本論文 20070823 對校內公開)

 

電子全文

        論文頁數:

78

摘要:

傳統上治療骨缺損主要使用移植的方式,但常仍無法達到理想的結果。目前新興的研究趨勢是利用組織工程(tissue engineering)治療的方式給予細胞,支架和促骨生成因子以促進骨癒合。近年更研發以基因治療(gene therapy)方式研究給予促骨生成因子以彌補蛋白質因子半衰期短之缺陷。活化基因基質(Gene-Activated MatrixGAM)技術是一種採納組織工程概念的非病毒性基因療法,活化基因基質是由支架與質體所組成,支架可容癒合細胞攀爬,而含有因子序列的質體在體內可進入細胞並表達所要因子。先前研究發現攜帶副甲狀腺素(parathyroid hormonePTH)基因的基因活化基因基質可促進長骨癒合時的軟骨內骨生成(endochondral bone formation),但對在顱顏骨之膜內骨生成(intramembranous bone formation)的作用仍屬未知。基於GAM技術很少被使用於牙科相關之顱顏骨缺損,所以進行本先驅研究以奠定未來應用活化基因基質技術於顱顏骨缺損的基礎。

 

    在細胞培養實驗中,發現帶有標記基因 (β-galactosidase)的質體可分別轉染人類牙齦纖維母細胞(gingival fibroblast)及胚胎腎細胞(HEK293),由X-gal染色和ortho-nitrophenyl-β-D- galactopyranosideONPG)酵素呈色分析發現質體可在細胞內能順利表達出標記蛋白活性。在體內實驗方面,發現膠原蛋白基質在大鼠頭蓋骨缺損中可隨時間降解,而在7-14天中消失不見。之後大量製備質體與膠原蛋白混合製備成活化基因基質並進行體內實驗,以X-gal染色發現帶有標記基因的活化基因基質在大鼠頭蓋骨缺損中可表達標記蛋白活性至少25天之久。最後建構出帶有副甲狀腺素基因的質體,確認序列無誤,並證實轉染該質體後細胞可順利表達副甲狀腺素mRNA

 

    本研究不但證實活化基因基質在顱顏骨缺損處可持續表達內含標記基因,且建構了未來可用於測試骨再生作用的含PTH質體,為後續使用活化基因基質於顱顏骨再生的研究奠下了良好基礎。

   
 

Bone grafting has been used commonly for treating osseous defects. However, the optimal healing is often difficult to achieve. Tissue engineering is an emerging strategy for treating osseous defects which can promote bone regeneration by providing appropriate cells, scaffolds and osteogenic factors. Recently, gene therapy has been advocated for prolong delivery of osteogenic factors to overcome the limitation of short half life for most protein factors. Gene–activated matrixGAMtechnology is a nonviral gene therapy which was developed in accordance with the concept of tissue engineering. A GAM is consisted of a matrix and a plasmid encoding gene of desired factors. The matrix facilitates ingrowth of cells during healing. The plasmid is responsible for expressing the desired factors after uptake by cells in vivo. Previous studied indicated that GAM encoding parathyroid hormone PTHsuccessfully promoted endochondral bone formation during healing of long bone osseous defects. However, its effects on intramembranous bone formation in craniofacial bone are largely unexplored. The present preliminary study was performed in order to set the basis for the application of GAM in treating craniofacial osseous defects.

 

The in vitro results of X-gal staining and ortho-nitrophenyl-β-D-galactopyranoside ONPGassay revealed that the marker geneβ-galactosidasein plasmid can be expressed with function both in transfected human gingival fibroblasts and embryonic renal cells HEK293. In vivo data indicated that collagen matrix degraded overtime and completely disappear between 7-14 days in rat calvarial defect. GAM marker gene study was performed using rat calvarial defect model. The X-gal staining demonstrated the existence of β-galactosidase activity at least 25 days in vivo. Finally, a plasmid containing PTH gene was constructed and sequence verified. In addition, the mRNA of PTH can be detected by RT-PCR in cells transfected with the constructed plasmid.

 

In this study, sustained expression of the marker gene in GAM can be achieved was demonstrated in craniofacial osseous defects. In addition, a plasmid containing PTH gene has been constructed for future GAM studies for bone regeneration. The results of this study have provided a valuable basis for future application of GAM technology in craniofacial bone regeneration.