免疫磁珠法分离和提纯雪旺细胞的实验研究  

Experimental study of separation and purification of Schwann cells by immunomagnetic heads method

作　　者：黄明[1] 罗卓荆[1] 肖伟[1] 张强[2] 

机构地区：[1]第四军医大学西京医院全军骨科研究所,西安710032 [2]解放军总院骨科,北京

出　　处：《中国矫形外科杂志》2008年第8期599-601,612,共4页Orthopedic Journal of China

基　　金：国家863高技术研究发展计划资助项目(No2002AA216101)

摘　　要：[目的]探讨利用免疫磁珠从SD大鼠坐骨神经分离培养获得大量、高纯度雪旺细胞的方法。[方法]选用4—7d SD大鼠，无菌条件下取双侧坐骨神经，解剖镜下剥离去除神经外膜，获得神经束，将神经束剪碎至1mm^3大小。采用双酶2次消化法消化，胎牛血清中止消化后，离心并加入DF12培养液培养。7d后应用免疫磁珠法对细胞进行纯化培养，培养2d后进行传代接种。培养过程中对细胞进行形态学观察、计数及活力测定；MTT法绘制细胞生长曲线，采用免疫细胞化学荧光染色对细胞进行鉴定并且计算所得细胞纯度。[结果]分离培养所得细胞即为雪旺细胞；采用免疫磁珠法能对培养所得雪旺细胞进行纯化。纯化后雪旺细胞活力为96％±0．5％，纯度98％±1．1％，培养2d后就可以传代。[结论]免疫磁珠法可以用于雪旺细胞的纯化培养，所得雪旺细胞纯度高，活力强，能满足组织工程人工神经的需要。[ Objective ] To introduce a method to obtain Schwann cells from newly born SD rats massively and purely by immunomagnetic heads method. [ Method] SD rats that had been born within 5 to 7 days were used. Their bilateral sciatic nerves were dissected under sterile condition. Under 16 × microscope the nerve fascicles and the epineurium were carefully extracted in oder to get the nerve tract without impurity cell. Then the nerve tract was cut into small particle about 1 mm^3. Two enzymes （0. 25% trypsinase and 0.2% collagenase Ⅰ ） were used to digest the particle of sciatic nerve specimens twice. After using 20% fetal bovine serum to stop the process of digestion, centrifugate （ 1000r/min,5 min） and DF12 culture medium was added into the precipitation. Seven days later, Schwann ceils were purified with immunomagnetic heads. During the whole process, the change of Schwann cells was observed under the inverted biological microscop and vital force of Schwann cells was evaluated. The growth curve of Sehwann cell was drawn with MTF. The Sehwann cells was identieated under immunofluoreseent test and record the purity. [Result]The cell separated from the SD rat＇s sciatic nerve and cultured in incubator was affirmed as Sehwann cells. Immunomagnetie heads can be used to purify the Sehwann cells. The 96% vigor and 98% pure Sehwann cell euhures were generated and passaged after two days. [ Conclusion ] This method can obtain massive purified normal sehwann cells to satisfy the need of tissue-engineered bioartifieial nerve graft.

关 键 词：雪旺细胞 免疫磁珠 坐骨神经 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]