研究生: |
余則威 Tse-Wei Yue |
---|---|
論文名稱: |
利用EDC/NHS 肝素化的小腸黏膜吸附第二型重組類腺病毒進行基因傳遞 EDC/NHS-Mediated Heparinization of Small Intestinal Submucosa for Recombinant Adeno-Associated Virus Serotype 2 Binding and Transduction |
指導教授: |
湯學成
Shiue-Cheng Tang |
口試委員: | |
學位類別: |
碩士 Master |
系所名稱: |
工學院 - 化學工程學系 Department of Chemical Engineering |
論文出版年: | 2007 |
畢業學年度: | 95 |
語文別: | 英文 |
論文頁數: | 37 |
中文關鍵詞: | 吸附 、基因表現 、基因傳遞 、肝素 、小腸下層黏膜 |
外文關鍵詞: | Adsorption, Fibroblast, Gene expression, Gene transfer, Heparin, SIS |
相關次數: | 點閱:2 下載:0 |
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使用基因載體作為治療工具的挑戰在於是否能控制在特定組織
局部釋放載體,一個可能的解決方法是植入帶有病毒的組織工程支
架,以達到局部基因傳遞的效果。在這篇研究中我們先將第二型重組
類腺病毒與肝素化小腸黏膜結合,再將細胞加到帶有病毒的小腸黏膜
上,細胞貼附生長後達到基因轉導的效果。肝素是第二型重組類腺病
毒的接受器,肝素化小腸黏膜的製備是利用交聯劑EDC 和NHS 將肝
素與小腸黏膜作交聯,肝素化後的小腸黏膜可用來吸附第二型重組類
腺病毒。從報導基因EGFP 和β-galactosidase 的表現顯示出肝素化小腸
黏膜所吸附的病毒具有活性,可以在培養細胞時達到基因傳遞的效
果。我們的研究顯示出經過化學修飾後的組織,也就是肝素化小腸黏
膜,具有吸附第二型重組類腺病毒和基因轉導的效果,所以肝素化小
腸黏膜可以做為局部基因傳遞一個很好的工具。
A major challenge in the use of gene transfer vectors as therapeutic tools is
controlling vector administration at a desired tissue site. One potential solution is
implanting tissue-engineering constructs loaded with gene transfer vectors such as
viruses for localized transgene delivery. In this work, we conjugated recombinant
adeno-associated virus serotype 2 (rAAV2) to a heparinized small intestinal
submucosa (H-SIS) matrix, which resulted in vector transduction upon cellular
adhesion. H-SIS was prepared by incorporating heparin, the rAAV2 receptor, into SIS
through N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) and
N-hydroxysuccinimide (NHS) mediated crosslinking. Incorporated heparin adsorbed
rAAV2 onto the H-SIS matrix for conjugation. Using green fluorescent protein and
□-galactosidase as reporters, we showed that conjugated rAAV2 was active and
capable of mediating transgene delivery in cell culture. Our work provides a unique,
modified tissue substrate H-SIS for rAAV2 binding and transduction, which can be a
useful tool in developing localized gene transfer.
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