聚乙烯乙烯醇薄膜接枝離胺酸與1,4-丁二胺對神經元細胞行為與功能性之影響

Abstract

本實驗是以乾式法將高分子乙烯乙烯醇(EVAL)聚合物製成薄膜之後，用化學改質的方式將表面氫氧基(-OH)接枝甲基丙烯醯氯(MAC)以間位氯化過氧苯酸(mCPBA)將烯烴雙鍵進行環氧化反應化後接枝離胺酸(L-lysine)單體(E-Lys)和1,4-丁二胺(E-14)。並且將接枝離胺酸(L-lysine)後的薄膜用碳化二亞胺(EDC)修飾離胺酸上的羧酸基接上14-丁二胺(E-Lys-14)。 之後利用體外細胞培養的方法，來探討材料與神經細胞之間的生物適合性，將改質前以及改質後的薄膜培養小鼠初代顆粒型神經細胞，以MTT測試來比較細胞的活性並且用光學顯微鏡和曠時攝影來觀察細胞貼附在接枝1,4-丁二胺、離胺酸以及經1,4-丁二胺修飾之離胺酸並和塗佈聚離胺酸(poly-D-lysine)的乙烯乙烯醇薄膜之細胞型態與行為。 另外再利用螢光顯微鏡來觀察神經細胞是否有發展出功能性之結構。最後利用西方點墨法與麩胺酸釋放來測量神經細胞在各材料上之蛋白質表現量與神經傳導功能之差異。 整體而言小鼠顆粒型神經細胞培養於離胺酸修飾1,4-丁二胺時(E-Lys-14)，不論是細胞活性、整體蛋白質表現，甚至是培養天期都優於EVAL塗佈聚離胺酸薄膜。接枝離胺酸修飾1,4-丁二胺的確具有取代塗佈聚離胺酸培養神經細胞的潛力。

We prepare poly(ethylene-co-vinyl alcohol)(EVAL) membranes by the dry processing ,and then grafted methacryloyl chloride (MAC)on the hydroxyl group of these membrane surface by the way of chemical modification. Furthermore, we grafted different diamines, lysine and modified lysine with 1,4-diaminobutane by EDC/NHS method on them. We cultured the cerebellar granule neurons of the Wistar rat on the modified membranes and compared with the relative activity by MTT assays. In addition, the cell morphology and behavior was observed by optical microscope and time-lapse. The functional structures of cerebellar granule neurons cultured on different membranes were observed by fluorescence microscope. We also used Western blot and glutamate release to mesure the protein expression and neural transmission. Overall, the modification of the EVAL with lysine modified 1, 4-diaminobutane ( E-Lys-14) have better relative activity, higher protein expression level and longer culture duration than coating PDL. E-Lys-14 may be the batter substrate to substitude coating PDL to culture neurons.