劉濤 1 , 陳艷芳 2 , 劉義 3
  • 四川大學(xué)華西醫(yī)院 1耳鼻咽喉科;2麻醉與危重急救研究實(shí)驗(yàn)室，四川成都 610041;3廣東省云浮市人民醫(yī)院耳鼻喉科，廣東云浮 527300;

目的：機(jī)械分離、培養(yǎng)小鼠耳蝸螺旋神經(jīng)元，并進(jìn)行免疫熒光細(xì)胞學(xué)鑒定，為后期進(jìn)一步的實(shí)驗(yàn)研究提供實(shí)驗(yàn)材料。方法：采用初出生1～5天以內(nèi)的昆明小鼠進(jìn)行解剖、機(jī)械分離以獲得螺旋神經(jīng)節(jié)組織，進(jìn)行原代培養(yǎng)后，應(yīng)用神經(jīng)微絲蛋白（Neurofilament protein,NFP－H）單克隆抗體進(jìn)行免疫熒光細(xì)胞學(xué)鑒定。結(jié)果：機(jī)械分離后獲得的螺旋神經(jīng)節(jié)組織中的螺旋神經(jīng)元，在體外培養(yǎng)條件下可以存活并進(jìn)行正常分化。典型的螺旋神經(jīng)元，其細(xì)胞形態(tài)呈橢圓形,胞體透明光滑、接近生理形態(tài)。熒光染色標(biāo)記后，胞體和神經(jīng)突起均顯色好，Schwann細(xì)胞和成纖維細(xì)胞未著色。結(jié)論：應(yīng)用機(jī)械分離的方法獲得小鼠耳蝸螺旋神經(jīng)節(jié)組織并進(jìn)行培養(yǎng)，耳蝸螺旋神經(jīng)元在體外可以穩(wěn)定地存活生長(zhǎng)。培養(yǎng)獲得的細(xì)胞形態(tài)和生存狀態(tài)接近生理狀態(tài)，滿足電生理、免疫細(xì)胞化學(xué)、藥理學(xué)等研究。應(yīng)用特異性的神經(jīng)微絲蛋白對(duì)培養(yǎng)獲得的螺旋神經(jīng)元進(jìn)行免疫熒光細(xì)胞學(xué)鑒定，特異性好，熒光顯色好。

引用本文： 劉濤,陳艷芳,劉義. 小鼠耳蝸螺旋神經(jīng)元的原代培養(yǎng)及鑒定. 華西醫(yī)學(xué), 2008, 23(1): 104-105. doi: 復(fù)制

1. Bianchi LN,Cohan CS.Developmental regulation of neurite-promoting factorinfliuencing statoacoustic neurons[Ｊ].Devel Brain Res,1991,64:167－74..
2. 李剛，唐癑秌，程薇波．應(yīng)用LsAB技術(shù)顯示五羥色胺受體亞型1A在小鼠螺旋神經(jīng)元上的分布[Ｊ]．聽(tīng)力學(xué)及言語(yǔ)疾病雜志，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. 黃世楷，等，譯．神經(jīng)元形態(tài)與功能[Ｍ]．上?？萍汲霭嫔纾?986:6．.
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. 徐定遠(yuǎn)，劉世喜，劉進(jìn)．內(nèi)皮素1在豚鼠耳蝸螺旋神經(jīng)節(jié)的分布[Ｊ]．臨床耳鼻咽喉頭頸外科雜志，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. 孫建軍，劉鋌．螺旋神經(jīng)元原代培養(yǎng)及其免疫細(xì)胞化學(xué)鑒定[Ｊ]．中華耳鼻咽喉科雜志，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.應(yīng)用膜片鉗技術(shù)對(duì)螺旋神經(jīng)元放電類型的初步研究[Ｊ]．聽(tīng)力學(xué)及言語(yǔ)疾病雜志，2002，10（4）：250-252．.
  1. 1. Bianchi LN,Cohan CS.Developmental regulation of neurite-promoting factorinfliuencing statoacoustic neurons[Ｊ].Devel Brain Res,1991,64:167－74..
  2. 2. 李剛，唐癑秌，程薇波．應(yīng)用LsAB技術(shù)顯示五羥色胺受體亞型1A在小鼠螺旋神經(jīng)元上的分布[Ｊ]．聽(tīng)力學(xué)及言語(yǔ)疾病雜志，2007，15（3）：235-237．.
  3. 3. Harrison RG.Observation on the living developing nerve fiber[Ｊ].Ant Rec，1907,1:116．.
  4. 4. Nakai J.Dissociated dorsal root ganglia in tissueculure[Ｊ].Am J Anat,1956,99(1):81．.
  5. 5. 黃世楷，等，譯．神經(jīng)元形態(tài)與功能[Ｍ]．上?？萍汲霭嫔纾?986:6．.
  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. 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. 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. 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. 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. 11. 徐定遠(yuǎn)，劉世喜，劉進(jìn)．內(nèi)皮素1在豚鼠耳蝸螺旋神經(jīng)節(jié)的分布[Ｊ]．臨床耳鼻咽喉頭頸外科雜志，2007，21（4）：176-177．.
  12. 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. 13. 孫建軍，劉鋌．螺旋神經(jīng)元原代培養(yǎng)及其免疫細(xì)胞化學(xué)鑒定[Ｊ]．中華耳鼻咽喉科雜志，1998，33（4）：196－198．.
  14. 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. 15. Ross MD.Multipolar neurons in the spiral ganglion of rat[Ｊ].Acta otolaryngol,1973,76:381-394..
  16. 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. 17. Sobkowicz HM,Loftus JM,slapnick,et al.Tissue culture of the organ of corti[Ｊ].Acta Otolaryngol,1993,502:3-36..
  18. 18. 唐癑秌，Davis Robin.應(yīng)用膜片鉗技術(shù)對(duì)螺旋神經(jīng)元放電類型的初步研究[Ｊ]．聽(tīng)力學(xué)及言語(yǔ)疾病雜志，2002，10（4）：250-252．.