Primary Culture in Vitro and Identification of Murine Spiral Ganglion Neurons_West China Medical Journal

Authors：

LIU Tao ¹ , CHEN anfang ² , LIU Yi ³

Department of Otolaryngology,West China Hospital,Sichuan University,Chengdu 610041,China;

Corresponding author：

Keywords：

螺旋神经元; 原代培养; 神经微丝蛋白; 免疫荧光细胞学

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目的：机械分离、培养小鼠耳蜗螺旋神经元，并进行免疫荧光细胞学鉴定，为后期进一步的实验研究提供实验材料。方法：采用初出生1～5天以内的昆明小鼠进行解剖、机械分离以获得螺旋神经节组织，进行原代培养后，应用神经微丝蛋白（Neurofilament protein,NFP－H）单克隆抗体进行免疫荧光细胞学鉴定。结果：机械分离后获得的螺旋神经节组织中的螺旋神经元，在体外培养条件下可以存活并进行正常分化。典型的螺旋神经元，其细胞形态呈椭圆形,胞体透明光滑、接近生理形态。荧光染色标记后，胞体和神经突起均显色好，Schwann细胞和成纤维细胞未着色。结论：应用机械分离的方法获得小鼠耳蜗螺旋神经节组织并进行培养，耳蜗螺旋神经元在体外可以稳定地存活生长。培养获得的细胞形态和生存状态接近生理状态，满足电生理、免疫细胞化学、药理学等研究。应用特异性的神经微丝蛋白对培养获得的螺旋神经元进行免疫荧光细胞学鉴定，特异性好，荧光显色好。

Citation： LIU Tao,CHEN anfang,LIU Yi,et al.. Primary Culture in Vitro and Identification of Murine Spiral Ganglion Neurons. West China Medical Journal, 2008, 23(1): 104-105. doi: Copy

1.	Bianchi LN,Cohan CS.Developmental regulation of neurite-promoting factorinfliuencing statoacoustic neurons[Ｊ].Devel Brain Res,1991,64:167－74..
2.	李刚，唐癑秌，程薇波．应用LsAB技术显示五羟色胺受体亚型1A在小鼠螺旋神经元上的分布[Ｊ]．听力学及言语疾病杂志，2007，15（3）：235-237．.
3.	Harrison RG.Observation on the living developing nerve fiber[Ｊ].Ant Rec，1907,1:116．.
4.	Nakai J.Dissociated dorsal root ganglia in tissueculure[Ｊ].Am J Anat,1956,99(1):81．.
5.	黄世楷，等，译．神经元形态与功能[Ｍ]．上海科技出版社，1986:6．.
6.	Kimura K,Mat sumoto N,Kitada M,et al.Neurite out growth from hippocampal neurons is promoted by choroid plexus ependymal cells in vit ro[Ｊ].J Neurocytol,2004,33:465-476..
7.	Deumens R,Koopmans G C,Den Bakker C G,et al.Alignment of glial cells stimulates directional neurite growth of CNS neurons in vitro[Ｊ].Neuroscience,2004,125:591-604..
8.	Gallo F,Morale M C,Spina2Purrello V,et al.Basic fibroblast growth factor(bFGF)act s on both neurons and glia to mediate the neurot rophic effect s of ast rocytes on L HRH neurons in culture[Ｊ].Synapse,2000,36:233-253..
9.	Chakrabort ty S,Kitada M,Mat sumoto N,et al.Choroid plexus ependymal cells enhance neurite outgrowth f rom dorsal root ganglion neurons in vitro[Ｊ].J Neurocy2tol,2000,29:707-717..
10.	Zhiping Zhou,Qing Liu,and Robin L.Davis.Complex regulationof spiral ganglion neuron firing patterns by neurotrophin-3[Ｊ].The Journal of Neurosicence,August 17,2005-25(33):7558-7566..
11.	徐定远，刘世喜，刘进．内皮素1在豚鼠耳蜗螺旋神经节的分布[Ｊ]．临床耳鼻咽喉头颈外科杂志，2007，21（4）：176-177．.
12.	Hoorens A,Pipeleers D.Nicotinamide protents human beta cells against chemically induced necrosis,but not against cytokine induced apoptosis[Ｊ].Diabetologia,1999,42:55-59．.
13.	孙建军，刘铤．螺旋神经元原代培养及其免疫细胞化学鉴定[Ｊ]．中华耳鼻咽喉科杂志，1998，33（4）：196－198．.
14.	Han DT,Yamashita T,Harada N,et al.Calcium mobilization in isolated cochlear sprail ganglion cells of the guinea pigs[Ｊ].Acta Otol,1993,Suppl 506:26-29..
15.	Ross MD.Multipolar neurons in the spiral ganglion of rat[Ｊ].Acta otolaryngol,1973,76:381-394..
16.	Ard,MD,Morest DK,Hauger SH.Trophic interactions between the cochleovestibular ganglion of the chick embryo and its synaptic targets in cultures[Ｊ].Neurosicence,1985,16:151-170..
17.	Sobkowicz HM,Loftus JM,slapnick,et al.Tissue culture of the organ of corti[Ｊ].Acta Otolaryngol,1993,502:3-36..
18.	唐癑秌，Davis Robin.应用膜片钳技术对螺旋神经元放电类型的初步研究[Ｊ]．听力学及言语疾病杂志，2002，10（4）：250-252．.

1. Bianchi LN,Cohan CS.Developmental regulation of neurite-promoting factorinfliuencing statoacoustic neurons[Ｊ].Devel Brain Res,1991,64:167－74..
2. 李刚，唐癑秌，程薇波．应用LsAB技术显示五羟色胺受体亚型1A在小鼠螺旋神经元上的分布[Ｊ]．听力学及言语疾病杂志，2007，15（3）：235-237．.
3. Harrison RG.Observation on the living developing nerve fiber[Ｊ].Ant Rec，1907,1:116．.
4. Nakai J.Dissociated dorsal root ganglia in tissueculure[Ｊ].Am J Anat,1956,99(1):81．.
5. 黄世楷，等，译．神经元形态与功能[Ｍ]．上海科技出版社，1986:6．.
6. Kimura K,Mat sumoto N,Kitada M,et al.Neurite out growth from hippocampal neurons is promoted by choroid plexus ependymal cells in vit ro[Ｊ].J Neurocytol,2004,33:465-476..
7. Deumens R,Koopmans G C,Den Bakker C G,et al.Alignment of glial cells stimulates directional neurite growth of CNS neurons in vitro[Ｊ].Neuroscience,2004,125:591-604..
8. Gallo F,Morale M C,Spina2Purrello V,et al.Basic fibroblast growth factor(bFGF)act s on both neurons and glia to mediate the neurot rophic effect s of ast rocytes on L HRH neurons in culture[Ｊ].Synapse,2000,36:233-253..
9. Chakrabort ty S,Kitada M,Mat sumoto N,et al.Choroid plexus ependymal cells enhance neurite outgrowth f rom dorsal root ganglion neurons in vitro[Ｊ].J Neurocy2tol,2000,29:707-717..
10. Zhiping Zhou,Qing Liu,and Robin L.Davis.Complex regulationof spiral ganglion neuron firing patterns by neurotrophin-3[Ｊ].The Journal of Neurosicence,August 17,2005-25(33):7558-7566..
11. 徐定远，刘世喜，刘进．内皮素1在豚鼠耳蜗螺旋神经节的分布[Ｊ]．临床耳鼻咽喉头颈外科杂志，2007，21（4）：176-177．.
12. Hoorens A,Pipeleers D.Nicotinamide protents human beta cells against chemically induced necrosis,but not against cytokine induced apoptosis[Ｊ].Diabetologia,1999,42:55-59．.
13. 孙建军，刘铤．螺旋神经元原代培养及其免疫细胞化学鉴定[Ｊ]．中华耳鼻咽喉科杂志，1998，33（4）：196－198．.
14. Han DT,Yamashita T,Harada N,et al.Calcium mobilization in isolated cochlear sprail ganglion cells of the guinea pigs[Ｊ].Acta Otol,1993,Suppl 506:26-29..
15. Ross MD.Multipolar neurons in the spiral ganglion of rat[Ｊ].Acta otolaryngol,1973,76:381-394..
16. Ard,MD,Morest DK,Hauger SH.Trophic interactions between the cochleovestibular ganglion of the chick embryo and its synaptic targets in cultures[Ｊ].Neurosicence,1985,16:151-170..
17. Sobkowicz HM,Loftus JM,slapnick,et al.Tissue culture of the organ of corti[Ｊ].Acta Otolaryngol,1993,502:3-36..
18. 唐癑秌，Davis Robin.应用膜片钳技术对螺旋神经元放电类型的初步研究[Ｊ]．听力学及言语疾病杂志，2002，10（4）：250-252．.

West China Medical Journal

Primary Culture in Vitro and Identification of Murine Spiral Ganglion Neurons

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