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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。
Objective: To explore the method of using immunomagnetic beads to isolate and culture large numbers of high purity Schwann cells from the spiral ganglion of newborn SD rats. Methods: 1-3d SD rats were selected and exposed to the blisters on both sides under aseptic conditions. The scrolls were carefully dissected at high magnification, the cochlea were opened and the cochlear tissues were removed completely. The blood vessels and basilar membrane of the lateral walls of the membrane were removed and removed Membrane tissue, then cut. Digest with 0.25% trypsin, stop digestion with fetal bovine serum, and add DMEM / F12 culture solution after centrifugation. After 3-5 days, the cells were purified by immunomagnetic beads positive sorting method. After culturing for 2 days, the cells were passaged and inoculated. Cultured rat cochlear Schwann cells were observed morphologically and their growth curves were drawn. Immunofluorescence staining Cells were S-100 immunofluorescently identified and cell purity was calculated. Results: The cells obtained after isolation and culture were Schwann cells. Purified cells were purified by immunomagnetic beads and the purity of purified Schwann cells was 97% ± 1.2%. Conclusion: Immunomagnetic beads method is an effective method for the isolation and purification of spiral ganglion Schwann cells in neonatal rat offspring. The obtained cochlear Schwann cells have strong vitality and high purity, and can be used for the research on the growth and regeneration of Schwann cells in the cochlea and spiral ganglion.