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研究生： 王志豪
Zhi-Hao Wang
論文名稱： 利用肝素化的纖維素基質吸附第二型重組腺病毒並進行局部基因傳導
Application of Heparinized Cellulose Affinity Membranes in Recombinant Adeno-Associated Virus Serotype 2 Binding and Delivery
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： 2008
畢業學年度： 96
語文別： 英文
論文頁數： 39
中文關鍵詞： 第二重組腺病毒 、親和性層析法 、纖維素 、基因傳導 、肝素
外文關鍵詞： AAV2, affinity membrane chromatography, cellulose, gene transfer, heparin
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    • The microporous affinity membrane based on cellulose matrices offers minimal mass-transfer effects in membrane chromatography with low nonspecific adsorption. In this
    research, we tested a novel application of the microporous, heparinized cellulose matrices (H-CM) for their affinity toward recombinant adeno-associated virus serotype 2 (rAAV2,
    which uses heparan sulfate proteoglycans as the primary cellular receptor) to develop a controlled, substrate-mediated viral vector delivery. We conjugated rAAV2 to an
    epoxy-crosslinked heparin cellulose membrane, which led to vector transduction upon cellular adhesion. When adhered, the human HT-1080 fibroblasts exhibited proliferation
    kinetics similar to those on the standard polystyrene tissue-culture surface. Using green fluorescent protein and β-galactosidase as reporters, we showed that the heparin-bound rAAV2 particles remained active and that the rAAV2-heparin binding was reversible and capable of mediating transgene delivery in cell culture. In addition, we applied the affinity membrane to adsorb unpurified rAAV2 from the crude lysate of packaging cells via the ligand-receptor binding, avoiding the use of conventional ultracentrifugation or chromatography in preparation of infectious rAAV2 for transduction. Our work explores a
    new application of affinity cellulose matrices in substrate-mediated viral vector delivery, which can be a useful tool in developing protocols for localized gene transfer.

    中文摘要 ABSTRACT 謝誌 CONTENT LIST OF FIGURES CHAPTER 1: INTRODUCTION CHAPTER 2: LITERATURE REVIEWS □ 2.1 Gene therapy □ 2.2 Cellulose and its derivatives □ 2.3 Sartobind® membrane adsorbers CHAPTER 3: MATERIALS AND METHODS □ 3.1 Cell culture □ 3.2 Cellular adhesion and kinetics of cell proliferation on H-CM □ 3.3 Viral vector preparation □ 3.4 Kinetics of rAAV2 binding to H-CM □ 3.5 Immunogold-silver staining of rAAV2 particles binding with H-CM □ 3.6 H-CM-mediated rAAV2 transduction □ 3.7 Assays CHAPTER 4: RESULTS AND DISCUSSION □ 4.1 Gross and microscopic images of heparinized cellulose membrane (H-CM) □ 4.2 Cellular adhesion and proliferation on H-CM □ 4.3 Binding of rAAV2 to H-CM □ 4.4 Recombinant AAV2 transduction mediated by H-CM □ 4.5 Affinity adsorption of rAAV2 from the crude lysate of packaging cells for H-CM-mediated transduction CHAPTER 5: CONCLUSIONS REFERENCE APPENDIX: APPLICATION OF HEPARINIZED CELLULOSE AFFINITY MEMBRANES IN RECOMBINANT ADENO-ASSOCIATED VIRUS SEROTYPE 2 BINDING AND DELIVERY

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