大鼠耳蜗雪旺细胞的体外培养和纯化

目的:探讨利用免疫磁珠从新生SD大鼠耳蜗螺旋神经节分离培养获得大量、高纯度雪旺细胞的方法。方法:选用1-3d SD大鼠,无菌条件下暴露双侧听泡,在高倍镜下仔细剥离蜗壳,开放耳蜗,完整取出耳蜗组织,分离并且除去膜蜗管外侧壁的血管纹和基底膜组织,然后剪碎。用0.25%的胰蛋白酶消化,用胎牛血清中止消化,离心以后加入DMEM/F12培养液培养。3-5天后对细胞应用免疫磁珠阳性分选方法进行纯化,培养2天后进行传代接种,培养过程中对提纯后的大鼠耳蜗雪旺细胞进行形态学观察、并绘制其生长曲线,采用细胞免疫荧光染色对细胞进行S-100免疫荧光鉴定并且计算细胞纯度。结果:分离培养后所得的细胞即为雪旺细胞;利用免疫磁珠阳性分选法对培养所得的细胞进行纯化,纯化后的大鼠耳蜗雪旺细胞纯度为97%±1.2%。结论:免疫磁珠法是一种有效的分离纯化新生大鼠仔鼠耳蜗螺旋神经节雪旺细胞的方法。所得耳蜗雪旺细胞活力强、纯度高,可以用于耳蜗雪旺细胞与螺旋神经节轴突的生长和再生等相关研究。

Purification and Separation of Cochlear Schwann Cells by ImmunomagneticBeads Method*

Objective: To introduce a method to obtain Schwann cells from newly born SD rats massively and purely by immuno-magnetic beads method.Method: SD rats,which were born 1-3days,were chosen.Under sterile conditions,expose the bilateral auditory vesicles,dissect the snail shell at high magnification,open cochlea and take out the cochlea completely,isolate and remove stria vascu-laris and basement membrane on the lateral wall of membranous cochlear duct,and cut in pieces.0.25% trypsin and 0.2% collagenase I was used for digestion,FBS was used to suspend the digestion,and that was centrifuged with the addition of DF12 medium.After 3-5days,cells were purified using immunomagnetic microbeads,and cell passage was done after culturing for two days.During the whole process,the change of Schwann cells was observed under the inverted biological microscope and vital force of Schwann cells was evalu-ated.The growth curve of Schwann cell was drawn with MTT.The Schwann cells was identified under immunofluorescence test and record the purity.Result: The cell separated from the SD rat’s spiral ganglia and cultured in incubator was affirmed as Schwann cells.Im-munomagnetic beads can be used to purify the Schwann cells.The 97%±1.2% pure Schwann cell cultures were generated and passaged after two days.Conclusion: This method can obtain massive purified normal Schwann cells to permit a study of the molecular nature of interacting cochlear glia and auditory neurons.