Cloning and Analysis of the Gene Induced by Cyclic Stretch Stimuli

利用差异显示技术克隆了一个周期性机械刺激特异性诱导的基因。成骨细胞系MC3T3E.1在周期性机械刺激或静态下培养。提取两种不同来源的RNA并用作差异显示的模板。在5个片段中，只有一个片段在机械刺激培养的细胞中在北方印迹中显著高表达。该基因片段长度为206 bp。将该片段用作探针用于进一步分析。该基因的全长为7.0bp。我们测定了该基因3'端2.5kbp的DNA序列。同源性分析表明，该基因是一个新基因，属于膜结合受体家族成员，能穿透细胞膜7次。RNA印迹显示，该基因在心脏、肺、骨骼肌中表达，这些细胞暴露于周期性机械刺激。我们将该基因命名为KEYAKI（Kinetics Evoked and Kinetics Induced）基因。完整肌腱、新鲜撕裂肌腱和肌腱切断后4周分别对应生理水平的刺激、无刺激和恢复一定水平的刺激。结果表明，KEYAKI在跟腱切断后表达量增加，在跟腱愈合后表达量减少。

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.