臺灣博碩士論文加值系統

Forkhead box D3 (FoxD3) 是一種轉錄因子與神經脊標記基因，被報導其能將人類纖維母細胞重新編程為神經脊細胞。在此研究中，一種具生物相容性的新 熱敏感型聚胺酯水膠被合成，並以此 PU 水膠作為 3D 列印之生物墨水。將 FoxD3 質粒與人類纖維母細胞混入 PU 水膠中再經由針頭共同擠壓以達到細胞轉染效 果。以兩種 PU 水膠做比對，探討水膠的流變特性包含模數、凝膠時間及剪切力 對於轉染效率的影響，並算出對應的剪應率及剪切力，實驗結果以 PU1500 具有 較好的轉染效果。在剪切力~190 Pa 進行轉染下，基因表現結果顯示人類纖維母 細胞能重新編程為類神經脊幹細胞，同時具有不錯的細胞存活率。隨後進一步結 合 3D 列印機，探討不同剪切力對於轉染率及細胞長期增生的影響，並以列印支 架的方式轉染後進行 12 天神經誘導培養，西方墨點法觀察後續蛋白表現證明細 胞確實能經誘導分化為類神經細胞。此類神經結構有望在未來應用在客製化之神 經修復上。

3D bioprinting is a technique which enables the direct printing of biodegradable materials with cells into 3D tissue. So far there is no cell reprogramming in situ performed with the 3D bioprinting process. Forkhead box D3 (FoxD3) is a transcription factor and neural crest marker, which was reported to reprogram human fibroblasts into neural crest stem-like cells. In this study, we synthesized a new biodegradable thermoresponsive waterborne polyurethane (PU) gel as a bioink. FoxD3 plasmids and human fibroblasts were co-extruded with the PU hydrogel through the syringe needle tip for cell reprogramming. The rheological properties of the PU hydrogel including the modulus, gelation time, and shear thinning were optimized for the transfection effect of FoxD3 in situ. The corresponding shear rate and shear stress were examined. Results showed that human fibroblasts could be reprogrammed into neural crest stem-like cells with high cell viability during the extrusion process under an average shear stress ~190 Pa. We further translated the method to the extrusion-based 3D bioprinting, and demonstrated that human fibroblasts co-printed with FoxD3 in the thermo-responsive PU hydrogel could be reprogrammed and differentiated into a neural-tissue like construct at 14 days after induction. The neural-like tissue construct produced by 3D bioprinting from human fibroblasts may be applied to personalized drug screening or neuroregeneration.

目錄
口試委員會審定書#
誌謝I
摘要II
AbstractIII
目錄V
圖目錄VIII
表目錄IX
第一章 文獻回顧1
1.1. 水膠的介紹1
1.2. 水膠中的聚胺酯2
1.3. 可分解水膠於組織工程之應用3
1.4. 3D組織列印支架5
1.5. 細胞之重新編程6
1.6. 誘導細胞的應用7
1.7. 基因轉染之技術與方法9
1.8. 研究動機10
第二章 研究方法12
2.1. 研究架構12
2.2. DL型聚乳酸二元醇合成及純化14
2.2.1. DL型聚乳酸二元醇合成及純化14
2.2.2. DL型聚乳酸二元醇分子量測定15
2.3. 水性聚胺酯(Waterborne polyurethane, PU)合成15
2.3.1. 原料與成分設計15
2.3.2. 分子量1500 g/mol DL型聚乳酸之PU1500合成16
2.4. PU1500奈米粒子性質分析19
2.4.1. 動態光散射(Dynamic light scattering, DLS)分析19
2.4.2. 穿透式電子顯微鏡(transmission Electron Microscopy, TEM)分析19
2.4.3. 小角度X光散射(Small-angle X-ray light scattering, SAXs)分析19
2.5. PU1500之物化性質分析20
2.5.1. 衰減式全反射(Attenuated total reflection, ATR)傅立葉紅外光譜分析20
2.5.2. 熱性質分析20
2.5.3. X光繞射(X-ray diffraction, XRD)分析21
2.5.4. 流變性質分析21
2.6. 組織工程實驗22
2.6.1. 細胞培養22
2.6.2. PU水膠之擠壓轉染法22
2.6.3. FoxD3 flag免疫染色分析23
2.6.4. 細胞存活率測定24
2.7. 基因及蛋白表現分析24
2.7.1. RNA抽取24
2.7.2. 反轉錄 (reverse transcription) 及基因表現分析25
2.8. 3D列印系統27
2.8.1. 3D列印之擠壓轉染分析27
2.8.2. 細胞長期增生測定27
2.9. 西方墨點法(Western blots)蛋白表現分析28
2.10. 統計學分析28
第三章 實驗結果29
3.1. 水性聚胺酯物化性質鑑定29
3.1.1. DL型聚乳酸二元醇核磁共振分析29
3.1.2. 水性聚胺酯核磁共振分析29
3.1.3. PU1500奈米粒子基礎性質分析30
3.1.4. 水性聚胺酯衰退式全反射傅立葉紅外光譜分析30
3.1.5. 水性聚胺酯熱重性質分析31
3.1.6. 水性聚胺酯之結晶性分析31
3.1.7. 小角度X光散射分析32
3.1.8. 水性聚胺酯流變性質分析32
3.2. 水性聚胺酯與組織工程實驗33
3.2.1. 細胞轉染後之FoxD3轉染率33
3.2.2. 細胞擠壓轉染後之存活率34
3.2.3. 細胞轉染後之相關基因表現分析34
3.2.4. 細胞3D列印轉染後之相關基因表現分析35
3.2.5. 剪切力對細胞轉染率及增生影響之分析36
3.2.6. 西方墨點法之神經蛋白表現分析36
第四章 討論37
4.1. PDLLA diol 核磁共振分析37
4.2. 溫度及離子對PU1500奈米粒子性質分析37
4.3. PU膜浸泡medium前後之氫鍵分析38
4.4. PU熱性質與結晶性分析39
4.5. PU1500分散液濃度對奈米粒子間之影響40
4.6. PU1500之流變性質分析40
4.7. 細胞及FoxD3共同擠壓轉染下之結果分析41
4.8. PU擠壓轉染後之細胞存活性分析42
4.9. 幹性及神經相關基因表現43
4.10.剪切力對轉染率及增生率之影響之分析44
第五章 結論45
參考文獻46
圖目錄
圖2.1. 實驗架構圖13
圖2.2. PU1500水性聚胺酯合成結構示意圖18
圖3.1. DL型聚乳酸二元醇之1H-NMR圖譜55
圖3.2. PU1500聚胺酯之1H-NMR圖譜分析56
圖3.3. PU1500奈米粒子之TEM影像57
圖3.4. 溫度及不同介質對PU1500奈米粒子水環直徑關係圖58
圖3.5. 分析PU1500浸泡medium前後氫鍵比例之ATR-IR圖譜59
圖3.6. PU1500之TGA及DSC溫度曲線圖60
圖3.7. PU1500、PU2000及PCL100的各結晶比例及XRD圖譜61
圖3.8. PU分散液中粒子濃度的影響。PU1500分散液在(A)1% NaHCO3(B)10% medium中不同固含量(0.2~30%)的SAXS分佈圖62
圖3.9. 固含量25%之PU1500及PU2000在37°C下之動態流變圖。(A) PU1500 與(B) PU2000之彈性模數(G’)與耗損模數(G’’)。(C, D)PU水膠之複合模數及複合黏度。(E)剪應率對黏度的關係圖。63
圖3.10. 人類纖維母細胞與FoxD3的轉染效率。經PU1500與PU2000擠壓轉染後之(A) FoxD3 flag免疫螢光圖及(B) 轉染效率量化圖。比例尺為50 μm。 (C) GFP質體和人類纖維母細胞以相似的剪切應力與PU水凝膠共擠出轉染之螢光表現圖。比例尺為100 m。64
圖3.11. 細胞擠壓轉染後之存活率。(A)VB-48螢光表現圖。(B)培養72小時存活率。(C) 直接擠壓轉染與PolyFect轉染後擠壓之存活率。66
圖3.12. 細胞轉染後之神經脊及幹性相關基因表現圖67
圖3.13. 細胞轉染後續神經誘導分化表現之神經相關基因qPCR表現圖68
圖3.14. 水膠與細胞列印支架69
圖3.15. 3D列印轉染法與擠壓轉染法之基因表現量。(A)幹性及神經脊相關基因(B)分化後神經相關基因表現圖71
圖3.16. 不同剪切力對轉染細胞的影響。(A) 剪切力~40 Pa與~250 Pa轉染下FoxD3 flag免疫螢光表現。(B) CCK-8細胞長期增生率。(C) 剪切力對轉染效率與細胞存活率之量化圖。72
圖3.17. 西方墨點法之神經蛋白表現74
表目錄
圖2.1. 實驗架構圖13
圖2.2. PU1500水性聚胺酯合成結構示意圖18
圖3.1. DL型聚乳酸二元醇之1H-NMR圖譜55
圖3.2. PU1500聚胺酯之1H-NMR圖譜分析56
圖3.3. PU1500奈米粒子之TEM影像57
圖3.4. 溫度及不同介質對PU1500奈米粒子水環直徑關係圖58
圖3.5. 分析PU1500浸泡medium前後氫鍵比例之ATR-IR圖譜59
圖3.6. PU1500之TGA及DSC溫度曲線圖60
圖3.7. PU1500、PU2000及PCL100的各結晶比例及XRD圖譜61
圖3.8. PU分散液中粒子濃度的影響。PU1500分散液在(A)1% NaHCO3(B)10% medium中不同固含量(0.2~30%)的SAXS分佈圖62
圖3.9. 固含量25%之PU1500及PU2000在37°C下之動態流變圖。(A) PU1500 與(B) PU2000之彈性模數(G’)與耗損模數(G’’)。(C, D)PU水膠之複合模數及複合黏度。(E)剪應率對黏度的關係圖。63
圖3.10. 人類纖維母細胞與FoxD3的轉染效率。經PU1500與PU2000擠壓轉染後之(A) FoxD3 flag免疫螢光圖及(B) 轉染效率量化圖。比例尺為50 μm。 (C) GFP質體和人類纖維母細胞以相似的剪切應力與PU水凝膠共擠出轉染之螢光表現圖。比例尺為100 m。64
圖3.11. 細胞擠壓轉染後之存活率。(A)VB-48螢光表現圖。(B)培養72小時存活率。(C) 直接擠壓轉染與PolyFect轉染後擠壓之存活率。66
圖3.12. 細胞轉染後之神經脊及幹性相關基因表現圖67
圖3.13. 細胞轉染後續神經誘導分化表現之神經相關基因qPCR表現圖68
圖3.14. 水膠與細胞列印支架69
圖3.15. 3D列印轉染法與擠壓轉染法之基因表現量。(A)幹性及神經脊相關基因(B)分化後神經相關基因表現圖71
圖3.16. 不同剪切力對轉染細胞的影響。(A) 剪切力~40 Pa與~250 Pa轉染下FoxD3 flag免疫螢光表現。(B) CCK-8細胞長期增生率。(C) 剪切力對轉染效率與細胞存活率之量化圖。72
圖3.17. 西方墨點法之神經蛋白表現74
表目錄
表2.1. 分子量1500 g/mol之20 mol%DL型聚乳酸組成之水性聚胺酯配方表16
表2.2. 各基因引子序列表26
表2.3. 神經分化液成分表26
表3.1. PU1500與PU2000分子量及奈米粒子基礎性質分析75
表3.2. PU1500與PU2000之熱裂解溫度及熔點75

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