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Cloning and Analysis of the Gene Induced by Cyclic Stretch Stimuli

循环拉伸刺激诱导基因的克隆与分析

基本信息

批准号：
11671418
负责人：
WADA Yuichi
金额：
$ 1.41万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

A gene specifically induced by cyclic mechanical stimuli was cloned using differential display. Osteoblastic cell-line MC3T3E.1 was cultured under cyclic mechanical stimuli or statically. RNA of two different sources were extracted and used as the templates for differential display. Only one fragment was significantly highly expressed in cells cultured with mechanical stimuli in northern blot out of 5 fragments. The length of gene fragment was 206 base pairs. This fragment was used for the probe for further analysis. Full length of this gene found out to be 7.0bp. We determined the DNA sequence of this gene up to 2.5 kbp from 3' terminus. Homology search revealed this gene was a novel gene and a member of cell membrane bound receptor that penetrates cell membrane 7 times. RNA blot revealed this gene was expressed in the heart, lung, skeletel muscles where cells were exposed to cyclic mechanical stimuli. We named this gene KEYAKI (Kinetics evoked and kinetics induced) gene.We examined in vivo pattern of expression using torn Achilles tendon rabbit. Intact tendon, fresh torn tendon and 4 weeks after tendon cut correspond to physiological level of stimuli, no stimuli, and recovered some level of stimuli respectively. The amount of expression was examined by semiquantitative RT-PCR.As a result, expression level of KEYAKI increased after the Achilles tendon was cut and decreased after the completion of healing of tendon.

利用差异显示技术克隆了一个周期性机械刺激诱导的基因。成骨细胞系MC3T3E.1在周期性机械刺激和静态培养条件下培养。提取两种不同来源的RNA作为差异显示的模板。在5个片段的Northern印迹中，只有1个片段在机械刺激培养的细胞中显著高表达。基因片段全长为206个碱基对。将此片段用于探针进行进一步分析。该基因全长为7.0bp。我们测定了该基因3‘端长达2.5kbp的DNA序列。同源性搜索表明，该基因是一个新基因，是细胞膜结合受体的一员，可穿透细胞膜7次。RNA印迹显示，该基因在心脏、肺和骨骼肌中表达，这些细胞暴露在周期性机械刺激中。我们将该基因命名为Keyaki(运动学诱发和动力学诱导)基因。我们用撕裂的跟腱兔检测了其在体内的表达模式。完整肌腱、新鲜撕裂肌腱和肌腱切断后4周分别对应生理刺激水平、无刺激和恢复一定水平的刺激。半定量RT-PCR检测Keyaki基因的表达，结果显示，跟腱切断后Keyaki基因表达水平升高，肌腱愈合完成后Keyaki基因表达水平降低。