A Study on Chitosan Nerve Conduit Loading aFGF Microsphere

神經導管接合術是利用顯微手術將神經兩斷端縫入生醫材料所製備之神經導管以橋接神經斷裂的兩端，神經可藉由軸突的再生而重新連接缺口兩端的神經使功能恢復，導管可減少神經拉扯時造成的張力並引導軸突的再生，亦可承載神經生長因子促進神經再生，此外，也可以將阻礙神經再生的細胞及其分泌物阻擋在管外。本研究引入了酸性纖維母細胞生長因子作為誘導神經再生之因子，以聚乳酸微小球作為酸性纖維母細胞生長因子的藥物釋系統，幾丁聚醣做為製作神經導管之材料，並以冷凍乾法與蘸濕浸泡法製備出含有兩種不同結構高分子塊材，再將高分子塊材捲製成特殊多層的導管，製作具有多層孔隙之新型神經再生導管。驗中，以SDS膠體電泳法尋求最佳包覆酸性纖維母細胞生長因子過程中的最佳穩定性條件。利用牛血清蛋白作為模組藥求得微小球的釋放模式。並於3T3纖維母細胞的活性實驗中求得最佳劑量。由ELSA試驗中映證酸性纖維母細胞生長因子能通過包覆過程並於微小球中釋放出。於掃描式電子顯微鏡觀察幾丁聚醣神經導管結構。驗結果顯示：在有機相的選擇上使用乙酸乙酯較二氯甲烷為佳，並於酸性纖維母細胞生長因子中添加聚離氨基酸有助於酸性纖維母細胞生長因子在包覆過程中保持其活性。利用牛血清蛋白的釋放實驗可知道所製備之聚乳酸微小球可保持14天以上的持續釋放。由3T3纖維母細胞的活性實驗中得知最佳的細胞活性劑量為1-10 ng/ml。在對六小時及兩天之釋放樣品進行ELISA試驗求得樣品釋放濃度約為0.7 ng/ml。在電子顯微鏡觀察下可發現所製備之微小球能均勻散布在導管之多孔結構中，而利用冷凍乾燥法和醮濕浸泡所製備出之神經導管也確實具備外側無孔隙之膜結構與內側滿布100μm孔隙之多孔狀結構。Nerve bridging is suture a biomaterial-made conduit and to overpass the damaged nerve end to end with microsurgery. Peripheral nerve could be bridged between the proximal nerve and the distal stump to restore the function. Nerve conduits could eliminate tension at the healing site and induce the regeneration of axons. Nerve conduits also could permit neurobiological recovery to enhance neural regeneration and stop cells and their secretions from obstructing neural regeneration. In this study, we provide acid fiberblast growth factor to induce the nerve to be regenerated and use PDLLA microsphere for drug delivery system. Chitosan was used to fabricate the nerve conduit. Combining Freeze-Drying method and dipping in the wet soaking method prepare out the conduit containing two kinds structure , and then rolled the material to make nerve conduit with pore and multi-layered. n the experiment, analyze with SDS page method to know the most stable environment for capsule acid fiberblast growth factor. Use bovine serum albumin as model drug to find the release profile of PDLLA microsphere. And test the best dosage in the cell activity experiment of 3T3 fiberblast cell. Prove acid fiberblast growth factor can be released form the PDLLL microsphere in the ELSA test. Observe chitosan nerve conduit structure by the scanning electron microscope. n The experimental result , ethyl acetate is better at the choice of organic phase than methylene chloride, and add ploy-lysine can promote the stable of the acid fiberblast growth factor undergo double emulsion process. Use bovine serum albumin as model drug to find the release profile of PDLLA microsphere. The result indicate PDLLA microsphere can keep more than 14 day releasing. the best activity dosage of cell is 1-10 ng/ml in the cell activity experiment of 3T3 fiberblast cell.for the release sample of six hours and two days, about 0.7 ng/ml acid fiberblast growth factor can be inspected in ELISA test. We can find the PDLLA microsphere can be kept in the porous layer of the chitosan nerve conduit.Combining Freeze-Drying method and dipping in the wet soaking method can prepare out the conduit containing two kinds structure whose non-porous film layer and 100μm pore sponge layer.目錄誌 …………………………………………………………………………………… I要 ……………………………………………………………………………………IIbstract ……………………………………………………………………………… III錄 …………………………………………………………………………………… V目錄 ……………………………………………………………………………… VII一章 緒論 .............................................................................. 1一節 周邊神經的生理學.................................................................................. 1二節 周邊神經的損傷...................................................................................... 2三節 周邊神經的再生...................................................................................... 2四節 協助神經再生的方法.............................................................................. 3五節 神經導管種類及應用.............................................................................. 5六節 製備神經導管之方式.............................................................................. 5七節 幾丁聚醣.................................................................................................. 7八節 纖維母細胞生長因子(FGF).................................................................... 8九節 聚乳酸(PDLLA) ...................................................................................... 9十節 明膠(Gelatin) ......................................................................................... 11十一節 多重乳化法........................................................................................ 11二章 研究動機與目的 ........................................................ 15三章 實驗材料與方法 ........................................................ 16一節 實驗藥品................................................................................................ 16二節 實驗儀器................................................................................................ 18三節 實驗溶液配製........................................................................................ 19四節 實驗方法................................................................................................ 22一項 aFGF穩定性試驗----SDS PAGE ................................................... 22二項 雙乳化法PDLLA微小球製備與觀測 ........................................... 23三項 微小球藥物釋放實驗.................................................................... 24四項 aFGF微量定量分析方法----ELISA test ....................................... 24五項 aFGF 細胞活性分析-MTS test .................................................... 25六項 雙結構Chitosan 神經導管製作 ................................................... 26七項 Chitosan 神經導管結構之觀察 ................................................... 28四章 結果與討論 ................................................................ 29一節 aFGF穩定性試驗----SDS PAGE ........................................................... 29二節 雙乳化法PDLLA微小球製備與觀測 ................................................... 31三節 微小球藥物釋放實驗............................................................................ 32四節 aFGF 細胞活性分析-MTS test ............................................................ 33五節 ELISA TEST .......................................................................................... 34六節 Chitosan 神經導管結構之觀測 ........................................................... 36五章 結論 ............................................................................ 41六章 參考文獻 .................................................................... 4