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RESEARCH OF THE ROLE OF ID PROTEIN(THE INHIBITOR OF DIFFERENTIATION) IN DEVELOPMENT OF RAT COCHLEA

大鼠耳蜗发育中ID蛋白（分化抑制剂）作用的研究

基本信息

批准号：
15591825
负责人：
OZEKI Masashi
金额：
$ 2.24万
依托单位：
NAGOYA CITY UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Cochlear progenitor hair cell line are useful for studies of cellular specification, gene expression features, and signal transduction involved in development of hair cells. To obtain embryonic and postnatal cochlear progenitor hair cell lines, we immortalized primary cultures of sensorineural epitherial cells from otocysts on embryonic day 12(E12) and explants of organ of Corti tissues on postnatal day 5(P5). Primary cultures and explants were then tranduced by thr E6/E7 genes of human papilloma virus type 16, and were passed for > 50 passages and partial clonal cells were isolated by limiting dilution. The expression of neuronal, neural, epithelial, hair cell markers and important transcription factors were examined in these cell lines. These cell lines may represent progeniotor hair cells at the different stages of cochlear development.Another result is as described below.Inhibitor of differentiation (Id3) is an important transcription factor expressed in the cochlear tissue and progenitor hair cells. It is likely to be involved in cell-cycle progression and the proliferation of cochlear progenitor hair cells during the embryonic stage. To characterize the expression of Id3 in the developing cochlear tissue and explore its role in the growth and proliferation of progenitor hair cells, the expression of Id3 gene in the developing cochlear tissue was examined by means of molecular biological methods and progenitor hair cell line were studied using specific antisense oligonucleotides. The Id3 gene was expressed in the rapidly growing otocyst on embryonic day 12 and specifically in the fundamental structures of the cochlea, e.g. the organ of Corti, spiral ganglions and steria vasclaris, on postnatal day 1. Inhibition of Id3 gene expression with antisense oligonucleotides in cultured otocyst epithelial cells and OT 12p reduced DNA synthesis and cell-cycle progression, suggesting that Id3 participates in the proliferation of cochlear progenitor hair cells.

耳蜗祖毛细胞系对研究毛细胞发育过程中的细胞特征、基因表达特征和信号转导具有重要意义。为了获得胚胎和出生后耳蜗祖毛细胞系，我们在胚胎第12天（E12）对耳蜗囊的感觉神经上皮细胞进行了永生化培养，并在出生后第5天（P5）对耳蜗组织的器官进行了外植体培养。用16型人乳头瘤病毒E6/E7基因诱导原代培养和外植体，传代50代，限稀释分离部分克隆细胞。在这些细胞系中检测了神经元、神经细胞、上皮细胞、毛细胞标记物和重要转录因子的表达。这些细胞系可能代表耳蜗发育不同阶段的前体细胞毛细胞。另一个结果如下所述。分化抑制因子（Id3）是耳蜗组织和原毛细胞中表达的重要转录因子。它可能参与胚胎期耳蜗祖毛细胞的细胞周期进程和增殖。为研究Id3基因在发育耳蜗组织中的表达，探讨其在原耳蜗毛细胞生长和增殖中的作用，采用分子生物学方法检测了Id3基因在发育耳蜗组织中的表达，并利用特异性反义寡核苷酸对原耳蜗毛细胞系进行了研究。在胚胎第12天，Id3基因在快速生长的耳囊肿中表达，在出生后第1天，在耳蜗的基本结构中表达，如Corti器官、螺旋神经节和steria vasclaris。用反义寡核苷酸抑制培养耳蜗上皮细胞和OT 12p中Id3基因的表达，可减少DNA合成和细胞周期进程，提示Id3参与耳蜗祖毛细胞的增殖。