大鼠内耳前体细胞的增殖研究  

The Precursor Cells from the Inner Ear of SD Rats

作　　者：曾亮[1] 江红群[1] 

机构地区：[1]南昌大学第一附属医院耳鼻咽喉头颈外科,南昌330006

出　　处：《听力学及言语疾病杂志》2014年第5期500-506,共7页Journal of Audiology and Speech Pathology

基　　金：江西省自然科学基金(2007GZY1408)资助

摘　　要：目的探讨不同年龄及生长因子对大鼠内耳前体细胞体外增殖的影响。方法 1分离取出出生后1天(P1)、7天(P7)、14天(P14)、21天(P21)、30天(P30)、60天(P60)各天龄12只(24耳)SD大鼠的球囊斑、椭圆囊斑、Corti器的前体细胞,在无血清条件下进行单细胞悬浮培养,至第七天,在倒置相差显微镜下对所形成的细胞球进行计数;2取P1大鼠球囊斑、椭圆囊斑、Corti器高增殖细胞分为实验组和对照组,实验组分为四组,培养液分别添加表皮生长因子(epidermal growth factor,EGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)、白血病抑制因子(leukemia inhibitory factor,LIF),对照组不加生长因子,观察不同生长因子对细胞球数量的影响;3免疫荧光染色检测细胞球的表型特征。结果1P1SD大鼠内耳各器官单细胞表现为nestin阳性,培养7天后,可形成悬浮的细胞球,球体细胞免疫荧光表现为nestin和BrdU阳性;2耳蜗Corti器只在P1、P7可形成细胞球,而球囊斑、椭圆囊斑各天龄段均可形成细胞球;3培养液中添加EGF、bFGF、IGF-1组,培养形成的细胞球数量明显增多,与对照组比较,差异有显著统计学意义(P<0.01);添加LIF组则与对照组比较差异无统计学意义(P>0.05)。结论大鼠内耳球囊斑、椭圆囊斑以及耳蜗Corti器内有高增殖能力细胞,能表达神经干细胞的特性;随着鼠龄的增长,这种增殖能力总体表现为减退趋势;EGF、bFGF、IGF-1均可单独促进P1大鼠内耳前体细胞的增殖,而LIF无促增殖作用。Objective To investigate the influence on the proliferation of the precursor cells from the inner ear of SD rats by different ages and growth factors in vitro .Methods The organs of utricle or saccule or cochlear Corti were isolated from different days’ SD rats born on different days（P1 ,P7 ,P14 ,P21 ,P30 ,P60） .From 12 SD rats of different age ,with 24 ears ,precursor cells were dissociated and cultivated under non -adherent conditions as single cell .We then counted the number of cell spheres under inverted phase contrast microscope .The results were statisti-cally anayzed and the quantity change curve of the same organ in different age rats were described until the single cells were cultured for 7 days .The culture medium was supplemented with defferent growth factor to observe the effects of the grow th factor on the number of cell spheres .Immunofluorescence assays were conducted for phenotype characterization .Results ① Some newly isolated single cells ,labeled by nestin ,were from each inner ear organ of rats（P1） .During 7 days in culture ,cells kept proliferation and formed suspended spheres ,which were expressed Br-dU and nestin positive .② The cell spheres could only formed from each cochlear Corti of rats （P1 ,P7） .But cell spheres that formed from each utricle or saccule were observed in a single saccule culture medium of all rats （P1~P60） .③The number of cell spheres ,which were formed by the isolated cells from inner ear organs of single utricle or saccule or cochlear Corti ,increased obviously in the presence of EGF or bFGF or IGF -1 ,but were not obviously in LIF .Statistically significant differences were seen between control group and every experimental group including EGF group ,bFGF group ,IGF-1 group（P〈0 .01） ,but were not seen between control group and LIF group （P〉0 .05） .Conclusion The highly proliferative cells were present in the vestibular organ （utricle or saccule） and cochle-ar Corti of SD rats .They could express the characterization of neural

关 键 词：球囊斑 椭圆囊斑 CORTI器 前体细胞 生长因子 

分 类 号：R764.3[医药卫生—耳鼻咽喉科]