大鼠内耳前体细胞的增殖研究

The Precursor Cells from the Inner Ear of SD Rats

曾亮;江红群;

1:南昌大学第一附属医院耳鼻咽喉头颈外科

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。

关键词

球囊斑;椭圆囊斑;Corti器;前体细胞;生长因子

基金项目(Foundation):

江西省自然科学基金(2007GZY1408)资助

作者

曾亮;江红群;

参考文献

1 Matsui JI,Parker MA,Ryals BM,et al.Regeneration and replacement in the vertebrate inner ear[J].Drug Discov Today,2005,10:1307.

2 Jero J,Aarnisalo A.Future therapeutic options for inner ear disease[J].Duodecim,2011,127:848.

3 Li HW,Liu H,Heller S.Pluripotent stem cells from the adult mouse inner ear[J].Nature Medicine,2003,9:1293.

4 江红群,王正敏,沈云珍,等.新生大鼠耳蜗增殖细胞的培养鉴定及超微结构观察[J].中华耳鼻咽喉头颈外科杂志,2007,42:42.

5 Wei D,Yamoah EN.Regeneration of the mammalian inner ear sensory epithelium[J].Curr Opin Otolaryngol Head Neck Surg,2009,17:373.

6 Diensthuber M,Heller S.Characterization of stem cells derived from the neonatal auditory sensory epithelium[J].HNO,2010,58:1056.

7 Oshima K,Senn P,Heller S.Isolation of sphere-forming stem cells from the mouse inner ear[J].Methods Mol Biol,2009,493:141.

8 Oiticica J,Barboza-Junior LC,Batissoco AC,et al.Retention of progenitor cell phenotype in otospheres from guinea pig and mouse cochlea[J].J Transl Med,2010,8:119.

9 Boddy SL,Chen W,Romero-Guevara R,et al.Inner ear progenitor cells can be generated in vitro from human bone marrow mesenchymal stem cells[J].J Regen Med,2012,7:757.

10 Koehler KR,Mikosz AM,Molosh AL,et al.Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture[J].J Nature,2013,500:217.

11 李强,马蕾.增龄对兔角膜缘干细胞标志物ABCG2表达的影响[J].山西医药杂志,2009,38:608.

12 Fan M,Chen W,Liu W,et al.The effect of age on the efficacy of human mesenchymal stem cell transplantation after a myocardial infarction[J].Rejuvenation Res,2010,13:429.

13 Wang WX,Hu XY,Xie XJ,et al.Nerve growth factor induces cord formation of mesenchymal stem cell by promoting proliferation and activating the PI3K/Akt signaling pathway[J].Acta Pharmacol Sin,2011,32:1 483.

14 Keerl S,Gehmert S,Song YH.PDGF and bFGF modulate tube formation in adipose tissue-derived stem cells[J].Ann Plast Surg,2010,64:487.

15 Yu JM,Bunnell BA,Kang SK.Neural differentiation of human adipose tissue-derived stem cells[J].Methods Mol Biol,2011,702:219.

16 Puri G,Kumar K,Singh R,et al.Effects of growth factors on establishment and propagation of embryonic stem cells from very early stage IVF embryos and their characterization in buffalo[J].Int J Stem Cells,2012,5:96.

17 Kouroupi G,Lavdas AA,Gaitanou M,et al.Lentivirus-mediated expression of insulin-like growth factor-1promotes neural stem/precursor cell proliferation and enhances their potential to generate neurons[J].J Neurochem,2010,115:460.

18 Whitlon DS,Grover M,Tristano J,et al.Culture conditions determine the prevalence of bipolar and monopolar neurons in cultures of dissociated spiral ganglion[J].Neuroscience,2007,146:833.

19 Okano T,Xuan S,Kelley MW.Insulin-like growth factor signaling regulates the timing of sensory cell differentiation in the mouse cochlea[J].J Neurosci,2011,31:18104.

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曾亮江红群