本計畫將發展一可在時間上精準控制基因釋放的非病毒載體輸送系統。我們將利用疊層組裝技術固定基因於生物材料上來進行原位轉染。利用帶正電的聚合物幾丁聚醣，將DNA 以多雙層 (multi-bilayers) 的方式固定在材料表面。並監測其質量和厚度以確認組裝步驟和層數的關係。紅外光譜、接觸角及電位等用以確認高分子間的靜電可以將其穩定在材料表面。DNA 的釋放時間和濃度則是藉由改變 pH 值、離子濃度、溶劑、分子量及組裝的時間來加以控制。爲了避免一般疊層組裝時，DNA 會有早期的爆發釋放現象，我們計畫在原來的DNA 多雙層頂部覆以聚麩胺酸作為保護的負電惰性高分子層。並比較不同分子量的聚麩胺酸和幾丁聚醣來最佳化其保護作用。此外，利用不同層數的聚麩胺酸和幾丁聚醣來延遲DNA 釋放，將可了解層數和釋放時間點的關係。最後我們以此系統攜帶BMP-7 基因轉染胎盤幹細胞，並以即時PCR 檢測以及ELISA 檢測其表現量，並同時評估其生物相容性和毒性。至於骨母細胞因誘導所產生的分化程度將以各時期骨分化標誌物來評估。也將評估基質骨化的程度以確認利用基因療法可新生成骨組織。上述的實驗，皆會進行有或無惰性多雙層保護情況下的影響評估。以證實藉由此釋放模型可於特定時間控制細胞分化和組織再生。 In this study, we would like to develop a non-viral gene delivery model to temporally control gene release. Layer-by-layer assembly techniques will be applied to immobilize genes encoding specific bioactive factors for inducing cell differentiation and improving tissue development. We will use chitosan as positive polymer to stably immobilize DNA on biomaterials as multi-bilayers. The film weight and thickness will be monitored to determine the relationship between assembly steps and bilayer numbers. Contact angle, FT-IR spectrometry, and zeta potential assays will be used to confirm the electrostatic forces between polymer layers can be firmly maintained on material surfaces. By controlling the pH values, salt density, solvent, polymer molecular weight, and assembly time, we will be able to control the periods and concentrations of DNA release. To avoid DNA burst release during the early stage of administration which frequently happened in conventional layer-by-layer DNA delivery, we will develop a protective inert multi-bilayers coating on the top of the original DNA-assembled multi-bilayers. We will introduce poly (L-glutamic acid) (PGA) as inert negative polymer due to its biocompatibility and biodegradability. Fluorescent dyes will be used to labeled PGA and chitosan, and different molecular weight of PGA and chitosan will be coated to optimize the protection effects. Then the PGA and chitosan in different multi-bilayers will be used to delay the release of DNA. The DNA release will be quantified to determine relationship of bilayer numbers and the lag time. The integrity of released DNA will be qualified by electrophoresis. Finally, we will apply this delivery system to in situ transfect placenta-derived multipotent cells (PDMCs) on biomaterials with osteogenic factor gene, BMP-7. The biocompatibility and cytotoxicity of this gene delivery system will be assessed by MTT and LDH assays, respectively. The internalized BMP-7 genes will be quantified by quantitative real-time PCR, and the expressed BMP-7 will be examined by ELISA. The time sequence of BMP-7 expression will be determined with the level of mRNA encoding osteoblast differentiation markers. The mineralization level will also be assessed to determine the effect of DNA immobilization strategy on mineral tissue regeneration. These results will be compared between the multi-bilayer groups with or without the inert multi-bilayer protection. Through these assays we may determine the extent to which our in situ gene delivery method could temporally control cell differentiation and tissue regeneration in specific period. We believe that this interdisciplinary and perspective project should benefit the field of regenerative gene therapy, expand the depth and extent of the tissue engineering research, and increase the visibility of our country in sciences. 研究期間：10008 ~ 10107
