摘要
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 efficiency of ethylene diamine tetraacetic acid （EDTA） to purify SD Schwann cells. Methods Firstly, epineurium and perineurium of SD bilateral sciatic nerves were peeled off under high power microscope. Afterwards the nerves without epineurium and perineurium were digested using collagenase NB4 for 30min. Then the supernatant was discarded after centrifuge at 600g. The resuspended sciatic verve fragments were cultured in medium supplemented with nerve growth factor for 1 week to get pre-degenerated tissues. The primary Schwann cells were harvested after the digestion of pre-degenerated sciatic nerves tissues with trypsin. Sub-confluent cells were treated by EDTA-PBS for 3 minutes and observed under microscope. Then, the cell pellet was resuspended and cultured in Schwann cell medium for 2 days. The purity of the subconfluent Schwann cells treated with EDTA-PBS was demonstrated by immunofluorescence staining with P75. Results The purity of Schwann cells was up to 99% or so after twice purification by EDTA. Conclusion EDTA is an efficient and effective method to purify SD Schwann cells.
出处
《山东大学学报（医学版）》
CAS
北大核心
2013年第11期21-24,共4页
Journal of Shandong University:Health Sciences
