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目的探讨乙二醇-双-(2-氨基乙醚)四乙酸(EDTA)纯化成年大鼠许旺细胞的可行性。方法取SD成年大鼠的双侧坐骨神经,高倍显微镜下剥除神经外膜及束膜。复合胶原酶NB4消化30 min,600g离心去除上清液。加入含神经生长因子的培养液,置于细胞培养箱中预变性1周。胰蛋白酶消化预变性的神经获得原代细胞。培养至亚融合状态,EDTA-PBS溶液作用3 min,显微镜下观察许旺细胞的变化。离心去上清,加入许旺细胞培养液悬浮,种植于培养瓶,置于培养箱培养。培养至亚融合,P75免疫荧光法检测许旺细胞的纯度。结果通过两次EDTA法纯化,许旺细胞纯度已经达到99%以上。结论 EDTA可以高效纯化成年大鼠的许旺细胞。
Objective To investigate the feasibility of purification of Schwann cells from adult rats by ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid (EDTA). Methods Bilateral sciatic nerves of adult SD rats were dissected and the epineurium and tunicate were removed under high power microscope. Collagenase NB4 digestion 30 min, 600g centrifuged to remove the supernatant. Add medium containing nerve growth factor and place in a cell incubator for 1 week. Primary cells are obtained by trypsin digestion of pre-degenerated nerves. Cultured to sub-fusion state, EDTA-PBS solution for 3 min, observe the changes of Schwann cells under a microscope. Centrifuge to the supernatant, add Schwann cells culture suspension, planting in culture flask, placed in the incubator. Culture sub-fusion, P75 immunofluorescence detection of Schwann cells purity. Results purified by two EDTA method, Schwann cell purity has reached more than 99%. Conclusion EDTA can efficiently purify Schwann cells from adult rats.