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目的探讨螺旋神经节细胞(spiral ganglion cells,SGCs)体外生长规律及特点,并观察阳离子脂质体转染SGCs的情况。方法从出生3~4 d的SD大鼠耳蜗中分离螺旋神经节组织,消化后放在含10%胎牛血清的DMEM/F12中培养,第2 d换用含2%B27及5μmol/L阿糖胞苷的Neurobasal培养基纯化SGCs,取第4 d活性良好的细胞爬片后,通过免疫荧光法用NF-200抗体鉴定SGCs;另外用质粒pEGFP-C2联合Lipofectamine 2000转染SGCs,观察其转染效率及对细胞活性的影响。结果体外培养的SGCs胞体饱满透亮,折光性好,一般能存活2周左右,第3~7 d活性最好。细胞对NF-200染色阳性;约10%的SGCs能被Lipofectamine 2000转染,但小部分细胞轴突缩短,甚至漂浮起来。结论含B27的Neurobasal培养基能培养出活性良好的SGCs;脂质体和质粒pEGFP-C2能成功地转染SGCs,但转染效率较低,并在一定程度上影响细胞的活性。
OBJECTIVE: To investigate the in vitro growth rules and characteristics of spiral ganglion cells (SGCs) and to observe the transfection of cationic liposomes into SGCs. Methods Spiral ganglion was isolated from the cochlea of SD rats born 3 ~ 4 days after birth. After digestion, the cells were cultured in DMEM / F12 containing 10% fetal bovine serum. The cells were switched to 2% B27 and 5μmol / L The SGCs were purified by Neurobasal medium of CGC, SGCs were identified by immunofluorescence with NF-200 antibody on the 4th day, and transfected into SGCs by using pEGFP-C2 and Lipofectamine 2000 Dyeing efficiency and its effect on cell activity. Results The somatic cell bodies of SGCs cultured in vitro were full of translucency and good refractivity. They generally survived for about 2 weeks and had the best activity on days 3 to 7. Cells stained positive for NF-200; about 10% of SGCs could be transfected with Lipofectamine 2000, but a small part of axons shortened and even floated. CONCLUSION: Neurobasal medium containing B27 can culture SGCs with good activity. Liposome and plasmid pEGFP-C2 can be successfully transfected into SGCs, but the transfection efficiency is low, and to a certain extent, affect the cell activity.