Li Lishu, Ikezono Tetsuo, Sekine Kuwon, Shindo Susumu, Matsumura Tomohiro, Pawankar Ruby, Ichimiya Issei, Yagi Toshiaki
Department of Otorhinolaryngology, Nippon Medical School, Tokyo, Japan.
Acta Otolaryngol. 2010 Aug;130(8):868-80. doi: 10.3109/00016480903493766.
We have cloned guinea pig Coch cDNA and the sequence information will be useful for future molecular study combined with physiological experiments. Proper Coch gene expression appears to be dependent on the unique extracellular micro-environment of the inner ear in vivo. These results provide insight into the Coch gene expression and its regulation.
To characterize the guinea pig Coch gene, we performed molecular cloning and expression analysis in the inner ear and cultured fibrocytes of the spiral ligament.
The Coch cDNA was isolated using RACE. Cochlin isofoms were studied by Western blot using three different types of mammalian inner ear. The cochlear fibrocytes were cultured and characterized by immunostaining. Coch gene expression in the fibrocytes was investigated and the influence of cytokine stimulation was evaluated.
The full-length 1991 bp Coch cDNA that encodes a 553 amino acid protein was isolated. The sequence had significant homology with other mammals, and the sizes of the Cochlin isoforms were identical. In the cultured fibrocytes, Coch mRNA was expressed in a very small amount and the isoform production was different, compared with the results in vivo. Cytokine stimulation did not alter the level of mRNA expression or isoform formation.
我们已经克隆了豚鼠Coch cDNA，该序列信息将有助于未来结合生理学实验进行分子研究。Coch基因的正常表达似乎依赖于体内内耳独特的细胞外微环境。这些结果为深入了解Coch基因表达及其调控提供了依据。
为了对豚鼠Coch基因进行表征，我们在内耳和螺旋韧带培养的成纤维细胞中进行了分子克隆和表达分析。
使用RACE技术分离Coch cDNA。通过蛋白质免疫印迹法，利用三种不同类型的哺乳动物内耳研究Cochlin异构体。培养耳蜗成纤维细胞并通过免疫染色进行表征。研究成纤维细胞中Coch基因的表达，并评估细胞因子刺激的影响。
分离出了全长1991 bp的Coch cDNA，其编码一个553个氨基酸的蛋白质。该序列与其他哺乳动物具有显著同源性，且Cochlin异构体的大小相同。与体内结果相比，在培养的成纤维细胞中，Coch mRNA表达量非常少，且异构体产生情况不同。细胞因子刺激并未改变mRNA表达水平或异构体形成。
