内耳支持细胞促进羊水干细胞定向分化为神经元的可能调控机制研究  

Regulatory Mechanisms of Inner Ear Supporting Cell Induced Directional Differentiation of Amniotic Fluid Stem Cells Into Neurons

作　　者：宗凌[1] 姜鸿彦[2] ZONG Ling;JIANG Hongyan(Department of Otorhinolaryngology,Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,China;Department of Otorhinolaryngology,Hainan General Hospital,Haikou 570311,China)

机构地区：[1]广州医科大学附属第二医院耳鼻咽喉科,广州510260 [2]海南省人民医院耳鼻咽喉医院,海口570311

出　　处：《中华耳科学杂志》2022年第3期422-426,共5页Chinese Journal of Otology

基　　金：国家自然科学基金-青年项目(81700912),广东省自然科学基金-博士启动(2016A030310286),广东省基础与应用基础研究基金-区域联合基金-青年基金项目(2019A1515110158)。

摘　　要：目的小鼠内耳支持细胞能诱导羊水干细胞(amniotic fluid stem cells,AFS)定向分化为功能性神经元,但其可能的调控机制仍未明确。本研究从AFS接种密度、内耳支持细胞来源及细胞周期蛋白激酶抑制因子P27^(kip1)表达差异入手,研究上述因素与AFS神经元定向分化的关系。方法分别将不同密度AFS(2×10^(5)、4×10^(5)、6×10^(5)、8×10^(5)、10×10^(5)),接种在内耳支持细胞饲养层表面,观察不同密度对神经元分化突起数量和长度的影响;将AFS分别接种于基底膜和前庭组织来源的饲养层表面,比较内耳支持细胞P27^(kip1)表达差异及其对神经元分化的影响。结果AFS以高密度(10×10^(5))接种于饲养层能产生更多形态典型的神经元(20倍镜视野下,平均208±25.7条神经突起),相对于低密度(2×10^(5))接种(20倍镜视野下,平均41±12.3条神经突起)存在统计学差异(P<0.01);随着接种细胞数的增加,神经突起的数量急剧增多,而神经突起的长度基本保持不变(平均长度88±7.8μm)(P>0.05)。前庭来源饲养层促分化能力较基底膜强,两种饲养层细胞均表达内耳支持细胞标记物P27^(kip1),前庭P27^(kip1)表达量(79.8±8.0%)明显高于基底膜(46.9±9.7%)(P<0.01);内耳支持细胞饲养层P27^(kip1)表达量与分化所得的神经突起的数量和长度呈正相关。结论AFS接种密度以及内耳支持细胞P27^(kip1)表达水平可能共同参与调控AFS向神经元的定向分化。Objective Mouse inner ear supporting cells can induce differentiation of amniotic fluid stem cells(AFS)into neurons,but the regulatory mechanism is still unclear.The current research studied the relations between directional AFS cell differentiation and density of AFS cells,source of inner ear supporting cells and cyclin kinase inhibitor P27^(kip1).Methods AFS cells were inoculated at different densities(2×10^(5),4×10^(5),6×10^(5),8×10^(5),10×10^(5))on feeder layers derived from Corti’s organ or vestibular tissue to observe the number and length of neurites,and to compare effects of P27^(kip1)on neuronal differentiation.Results High AFS cell density(10×10^(5))produced more neurons(average 208±25.7 neurites under 20x field of view)as compared to low AFS cell density(2×10^(5))(average 41±12.3 neurites under 20x field view)(P<0.01).The number of neurites increased sharply as AFS cells increased,but not the length of neurites(average length 88±7.8μm)(P>0.05).Neuronal differentiation on feeder layers derived from vestibular tissue was greater than on feeder layers from Corti’s organ.Feeder layer cells from both Corti’s organ and vestibular tissue expressed P27^(kip1),but higher(79.8±8.0%)on feeder layers from vestibular tissue than on feeder layers from Corti’s organ(46.9±9.7%)(P<0.01).P27^(kip1)on feeder layer of inner ear supporting cells was positively correlated with the number and length of neurites from AFS cells.Conclusion Density of AFS cells and P27^(kip1)on inner ear supporting cells may be involved in the regulation of neuronal differentiation of AFS cells.

关 键 词：羊水干细胞 饲养层 内耳支持细胞 神经元分化 P27^(kip1) 

分 类 号：R764[医药卫生—耳鼻咽喉科]