摘要
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 growth characteristic of spiral ganglion cells（SGCs） in vitro and the EGFP efficiency transfection with Lipofectamine 2000.Methods SGCs were isolated from the cochlea of newborn rats（P3-P4）,then were cultured in DMEM/F12 medium containing 10% fetal bovine serum.On the 2nd day,the medium was replaced with neurobasal medium containing 2% B27 and 5μmol/L Ara-c to purify SGCs.On the 4th day,the SGCs with good activity were identified with primary antibody NF-200 by immunofluorescence,and SGCs were transfected with lipofectamine 2000 and plasmid pEGFP-C2.The transfection efficiency was evaluated and the activity of SGCs was observed.Results The SGCs bodies presented transparent with good refraction.SGCs were alive for about 2 weeks and showed the best activity from the 3rd to 7th day.And the cells were positive for NF-200 immunostaining.About 10% of the SGCs were transfected but small part of the cells showed neurite recovery,even floated and died.Conclusion SGCs grew well in neurobasal medium containing B27;SGCs could be successfully transfected by plasmid pEGFP-C2 and Lipofectamine 2000,but the transfection efficiency was low and the cell activity was affected to some degree.
出处
《听力学及言语疾病杂志》
CAS
CSCD
北大核心
2010年第5期462-465,共4页
Journal of Audiology and Speech Pathology
基金
国家自然科学基金资助项目(No.30672307)
