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

耳蜗祖毛细胞系是研究毛细胞发育过程中的细胞特性、基因表达特征和信号转导的有用细胞。为了获得胚胎和出生后耳蜗祖毛细胞系，我们将胚胎第12天(E12)的耳囊感觉神经上皮细胞原代培养和出生后第5天(P5)的Corti组织器官外植体永生化。将人乳头瘤病毒16型E6/E7基因导入原代培养物和外植体，传代50代，有限稀释法分离出部分克隆性细胞。检测神经、神经、上皮、毛细胞标志物和重要转录因子在这些细胞系中的表达。这些细胞系可能代表了耳蜗发育不同阶段的前体毛细胞。另一个结果如下。分化抑制因子(ID3)是一种重要的转录因子，在耳蜗组织和前体毛细胞中表达。在胚胎期，它可能参与细胞周期进程和耳蜗祖毛细胞的增殖。为研究ID3基因在发育中耳蜗毛细胞中的表达特征，探讨其在毛细胞生长增殖中的作用，采用分子生物学方法检测了ID3基因在耳蜗发育组织中的表达，并用特异性反义寡核苷酸技术研究了ID3基因在毛细胞系中的表达。ID3基因在胚胎第12天在快速生长的耳囊中有表达，出生后第1天在耳蜗器、螺旋神经节和血管小体等基本结构中有表达。用反义寡核苷酸抑制培养的耳囊上皮细胞和OT12P中ID3基因的表达降低了DNA合成和细胞周期进展，提示ID3参与了耳蜗祖毛细胞的增殖。

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.