大鼠螺旋神经节干细胞体外优化培养方法的研究  

Optimizing Culture of Rat Spiral Ganglion Stem Cells

作　　者：朱一丹 吴翠萍 金越凡 殷善开[1,2] 李春燕 ZHU Yidan;WU Cuiping;JIN Yuefan;YIN Shankai;LI Chunyan(Department of Otorhinolaryngology,Shanghai Jiaotong University Affiliated Sixth People’s Hospital,Shanghai 200233,China;Institute of Otorhinolaryngology,Shanghai Jiaotong University Shanghai 200233,China)

机构地区：[1]上海交通大学医学院附属第六人民医院耳鼻咽喉头颈外科,上海200233 [2]上海交通大学耳鼻咽喉研究所,上海200233

出　　处：《中华耳科学杂志》2023年第2期239-244,共6页Chinese Journal of Otology

基　　金：国家自然科学基金(82071042);国家自然科学基金国际合作项目(81720108010);上海市自然科学基金(20ZR1442300)。

摘　　要：目的探索贴壁法进行螺旋神经节(Spiral Ganglion,SG)干细胞培养,同时与传统悬浮培养法对比培养的SG干细胞形态、增殖效率及分化表型。方法取新生1天SD大鼠耳蜗的螺旋神经节位置,以悬浮和单层贴壁的方式进行培养,观察SG干细胞的形态学特点。通过活/死细胞染色和Annexin-V/PI双染法比较其生长状态。通过Nestin、Sox2、Ki67等免疫荧光染色及生长曲线测定等方法对其增殖能力进行比较分析。体外胎牛血清诱导SG干细胞分化,通过Tuj1免疫荧光染色鉴定分化表型并比较分化效率。结果两种培养方式均可获得状态良好的高纯度SG干细胞,免疫荧光染色和生长曲线测定结果提示贴壁培养的干细胞的增殖活性较悬浮培养的干细胞更强。诱导分化后,两种培养方式获得的SG干细胞均可分化为Tuj1染色阳性的双极神经元样细胞。结论本研究结果表明单层贴壁可培养出状态良好的SG干细胞,并且其增殖能力较悬浮培养的干细胞更强。贴壁培养的干细胞可被可成功诱导分化为神经元。提示单层贴壁方式可作为一种稳定的SG干细胞培养方法,从而为SG干细胞的相关研究提供重要技术支持。Objective The monolayer adherent method was used to culture spiral ganglion(SG)stem cells and compared to traditional suspension culture on cell morphology,proliferation and differentiation phenotype.Methods The cochlear spiral ganglion of SD rats was collected on postnatal day 1 and cultured using either suspension or monolayer adherent methods.Morphological characteristics of SG stem cells were examined using Annexin-V/PI double staining.Cell proliferation was assessed by immunofluorescence staining with Nestin,Sox2 and Ki67,as well as the growth curve.Fetal bovine serum was used to induce in vitro SG stem cells differentiation and the differentiation phenotype was identified by Tuj1 immunofluorescence staining.The differentiation rate was compared between the two culture methods.Results High-purity healthy looking SG stem cells were obtained with both culture techniques,although stronger SG stem cells proliferation was observed in monolayer adherent cultures than in suspension cultures as determined by immunofluorescence staining and growth curve.Exposure to fetal bovine serum led to differentiation of SG stem cells into anti-Tuj1 positive bipolar neuron-like cells in both types of cultures,with no significant difference in differentiation rate.Conclusion This study shows that monolayer adherent cultures can be used to produce healthy SG stem cells with stronger proliferation than suspension cultures.Adherent cultured SG stem cells can be successfully induced to differentiate into neurons.This technique provides highly stable SG stem cell cultures and is thus an important technical support for research relted to SG stem cells.

关 键 词：螺旋神经节干细胞 单层贴壁培养 悬浮培养 增殖 

分 类 号：R764[医药卫生—耳鼻咽喉科]