| 摘要: | 神經再生醫學中，當神經受損後，其斷端殘餘部分中的細胞會釋放出生長因子(growth factor, GF)促進神經細胞分化(differentiation)及增生(proliferation)，幫助神經纖維再生，但由於細胞所產生的生長因子隨著受損時間有明顯的減少，無法持續釋放達到修復神經的目的，因此研究生長因子的持續釋放及結合不同生長因子的修復特性則成了重要的課題。本實驗利用靜電紡絲(electrospinning)技術結合同軸紡嘴(co-axial spinneret)，以聚乳酸(poly(lactic acid), PLA)及製孔劑(porogen)混合作為外管成分，聚氧化乙烯(poly(ethylene oxide), PEO)及聚乙二醇(poly(ethylene glycol), PEG)混和作為內管芯層結構，將兩種生長因子分別利用雙層乳化法(double emulsion method)及直接添加的方式混在內管溶液中，製備成含不同生長因子之微孔化芯鞘型纖維薄膜，藉由調配外管溶液中聚乳酸與製孔劑重量濃度比例的不同來改變纖維薄膜表面之孔隙率(porosity)，以達到控制釋放之目的。不同的生長因子在神經再生修復中扮演不同的角色，本實驗選用兩種不同的生長因子：神經生長因子(nerve growth factor, NGF)是直接添加在內管溶液中，作用是提升細胞的存活率及延長軸突(axon)生長，使細胞數量及狀況趨於穩定；而腦衍生神經滋養因子(brain-derived neurotrophic factor, BDNF)則是利用乳化的方式包覆在聚乳酸-甘醇酸(poly(lactic-co-glycolic acid, PLGA)後，再混入內管溶液中，在細胞穩定後釋放出來，使神經細胞分化、軸突髓鞘再生。首先選用與生長因子有相同分子量的細胞色素(cytochrome c)來模擬生長因子的釋放特性。接著進行生長因子的活性測試，將含有生長因子之培養基經過靜電紡絲後與大鼠嗜鉻性瘤細胞(pheochromocytoma, PC12)進行培養，藉由誘導PC12細胞的特有神經突觸(neurite)生長，利用倒立式顯微鏡觀察其生長情況並以影像處理軟體(image J)分析其神經突觸長度來測試生長因子經由靜電紡絲實驗後於人體體溫下的活性效能。
After nerve injury the growth factors secreted by neural cells in the distal nerve stump can improve cell growth, proliferation, and differentiation, but this supportive action may not be sustained indefinitely due to an obvious decline with time in cellular production of growth factors. Therefore, a continuous controlled supply of growth factors becomes a major research concern. Different growth factors play different roles in nerve regeneration. In this research, by using electrospinning technique to fabricate the growth factors-containing microporous microtube array, through the co-axial spinneret, the core- shell fibers were collected on the rotating collector. For the purpose of promoting cell survival and neurite elongation, we added the growth factor which can stabilize the quantity and condition of cells in inner core solution; and the other growth factor encapsulated into poly(lactic-co- glycolic acid) (PLGA) by double emulsion method which would released after cell stablized could induce cell differentiation and remyelination. The morphologies of micro-porous core-shell fibrous membranes were observed by the scanning electron microscope (SEM). By controlling the weight ratio of PLA and porogen could change the porosity and pore size on the surface to achieve the purpose of controlled release. At release test, we chose cytochrome c as a model compound which has the same molecular weight with growth factors. First system was cytochrome c added directly in inner cores, there were significant differences in release rate of different PLA/PEG weight ratio. Within the first day, PLA/PEG-0%-10% released approximately 60%, and PLA/PEG-40% released over 80%. It represented that the porosity and pore size increased by porogen content resulted in the release rate raised. And the second system was the cytochrome c encapsulated in PLGA which were released to outside significantly at the eighth day. Then proceeded the cell’s viability test of the growth factors which cultured with PC12 cells and observed by inverted microscope. By using electrospinning technique to fabricate the growth factors-containing microporous microtube array, combining the different characteristics of growth factors to achieve the purpose of nerve regeneration. |
