黃義侑臺灣大學:醫學工程學研究所伍怡貞Wu, I-ChenI-ChenWu2010-05-182018-06-292010-05-182018-06-292009U0001-3007200909462700http://ntur.lib.ntu.edu.tw//handle/246246/183732近年的脂肪組織工程研究中指出欲重建脂肪組織需結合細胞、支架和生長因子三大要素。脂肪前驅細胞(preadipocytes)是在脂肪組織工程中最常用的細胞來源，因為此細胞可在體外大量增生後，經過特定生長因子誘導分化成為脂肪細胞。細胞一旦分化後就不會再增生，所以欲得到大量的成熟脂肪細胞，必須在分化前先加入生長因子促使脂肪前驅細胞快速增生。生和分化都需要加入特定的生長因子。在本實驗中發現0.5 ng/mL bFGF可以促使3T3-L1在體外增生，而根據實驗室過去的研究也發現insulin 和dexamethasone可以誘導細胞分化。為了要讓增生的藥物和分生的藥物分別在不同階段釋放，我們所製備的核殼粒子(core-shell particles)的內殼為PDLLA微球體可包覆insulin和dexamethasone;外殼為明膠可吸附bFGF。因此，核殼粒子會先釋放bFGF，再釋放insulin和dexamethasone。實驗結果發現核殼粒子的外殼可控制釋放bFGF，並且在MTS的結果發現控制釋放bFGF可更有效促使細胞增生。在動物實驗中，將核殼粒子和細胞分別與兩種支架:支架膠原蛋白/玻尿酸混合膠體和明膠微球體打入Balb/c老鼠背部，10天後組織切片觀察。結，我們可以成功製備明膠鍍附PDLLA的核殼粒子並且可做為二階段藥物釋放系統應用於脂肪組織工程。第一階段的控制釋放bFGF可促使3T3-L1增生in vitro。結合脂肪前驅細胞、支架和二階段藥物系統最為軟組織重建的替代物是有潛力的。Recent studies in adipose tissue engineering have indicated that in vivo adipogensis requires an appopriate cell source, scaffold and growth factors. Preadipocytes, precursor cells committed to the adipocyte lineage are the ideal cell source. They are capable of proliferating to obtain extensive cell numbers in vitro and differenting into mature fat cells. Once cells differentiate, they are not capable of replication. Therefore, in order to induce the formation of larger fat tissue, it is essential to increase the numbers of preadipocytes before differentiation. readipocytes proliferate and differentiate in the presence of the growth factors. In this study, we found that 0.5 ng/mL bFGF is able to accelerate cell proliferation in vitro, and insulin and dexamethasone enable preadipocytes to differentiate according to the previous study. In order to release two types of growth factor in two-stage, we develop a method to obtain the core-shell particles with insulin/dex in the PDLLA core and gelatin shell layer for bFGF sorption. Core-shell particles are prepared for the controlled release of bFGF and followed by insulin/dex. The proliferation ability of 3T3-L1 incorporated by core-shell particles containing bFGF in vitro is assessed by the MTS assay. The MTS result shows that the controlled release of bFGF increases the number of preadipocytes. By injecting two types of scaffold, collagen/HA and gelatin microspheres respectively, in combination with core-shell particles and preadipocytes into the back of the Balb/c mice, the adipogenesis at the subcutasneous implantation sites is evalutated histologically.In conclusion, PDLLA particles (core) coating with gelatin (shell) are successfully acheived and ued as the two-step controlled release system in the application of adipose tissue engineering. The first step controlled release of bFGF increases the proliferation rate of 3T3-L1. The combination of preadipocytes and two-step drug release system with scaffold has the potential for augmentation of adipose tissue.口試委員會審定書 I謝 II要 IIIbstract IV目錄 IX目錄 XI一章 序論 11.1 軟組織缺陷 11.2 脂肪組織工程 21.2.1 細胞 21.2.1.1 3T3-L1 41.2.2 支架 41.2.3 生長因子 51.2.3.1 鹼性纖維細胞生長素 61.3 藥物釋放系統與在組織工程的應用 71.4 乳化法 91.5 核殼結構粒子 111.6 生物可分解性材料 121.6.1 聚乳酸 131.6.2 膠原蛋白 141.6.3 明膠 151.6.3.1 明膠作為藥物釋放載體 161.6.4 玻尿酸 18二章 研究概述 192.1 目前軟組織重建的方法 192.2 研究假設 192.3 研究目的 20三章 實驗材料與方法 223.1 實驗藥品 223.2 實驗儀器 243.3 製備包覆胰島素及dexamethasone之聚乳酸微球體 253.4 製備沒有包覆藥物的聚乳酸微球體 263.5 去溶劑法製備明膠鍍附之聚乳酸微球體 273.6 Entrapment Method製備明膠鍍附之聚乳酸微球體 283.7 FITC-Gelatin製備 303.8 共軛焦顯微鏡觀測 313.9 界面電位測量儀 313.10 掃描式電子顯微鏡觀測 323.11 細胞活性測試 323.12 明膠鍍附之聚乳酸微球體吸附bFGF 333.13 bFGF在37ﾟC下的穩定性測量 333.14 bFGF在第一階段之控制釋放 343.15 bFGF微量定量分析方法 ELISA test 343.16 脂肪前驅細胞初代培養 363.17 觀察bFGF對於preadipocyte的作用 413.18 Oil Red O染色觀察細胞分化 413.20 觀察bFGF對於3T3-L1增生的程度 423.21 3T3-L1在有控制釋放系統的生長情況 433.22 膠體製備 443.23 動物實驗 45四章 研究結果與討論 484.1 去溶劑法製備FITC-明膠鍍附之聚乳酸微球體 484.2 Entrapment Method製備FITC-明膠鍍附之聚乳酸微球體 534.3 交聯劑濃度與明膠鍍附程度之比較 564.4 觀察明膠鍍附之微球體之表面電位 594.5 觀察明膠鍍附之微球體之表面結構 604.6 細胞活性測試 614.7 脂肪前驅細胞與bFGF之關係 624.8 3T3-L1與bFGF之關係 654.9 bFGF 在37ﾟC環境下的穩定性測量 694.10 bFGF在第一階段之控制釋放 714.11 3T3-L1在有控制釋放系統的生長情況 724.12 動物實驗 75五章 結論 81考文獻 82application/pdf11227463 bytesapplication/pdfen-US脂肪組織工程脂肪前驅細胞明膠聚乳酸核殼粒子鹼性纖維細胞生長素adipose tissue engineeringgelatinpolylactic acidmicrospherecore-shell particlebasic fibroblast growth factorthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/183732/1/ntu-98-R96548057-1.pdf