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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671418/
• 关键词: Mechnical stimuli; Mechano-receptor; Differential display; Cell membrane; Gene cloning; 伸張刺激; クローニング; メカノレセプター; 膜型レセプター

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.

使用差分显示来克隆特异性诱导的循环机械刺激的基因。成骨细胞系MC3T3E.1在环状机械刺激下或静态培养。提取了两个不同源的RNA，并用作差分显示的模板。仅在5个片段中用机械刺激培养的细胞中，只有一个片段高度表达。基因片段的长度为206个碱基对。该片段用于探针进行进一步分析。该基因的全长发现为7.0bp。我们从3'末端确定了该基因的DNA序列高达2.5 kbp。同源性搜索揭示了该基因是一种新型基因，也是细胞膜结合受体的成员，可穿透细胞膜7次。 RNA印迹揭示了该基因在心脏，肺，骨骼肌肉中表达，其中细胞暴露于环状机械刺激中。我们命名了这个基因Keyaki（动力学引起的动力学和动力学）基因。我们使用撕裂的跟腱兔在体内表达模式。完整的肌腱，新鲜的肌腱和肌腱切割后的4周对应于刺激的生理水平，没有刺激，并恢复了一定程度的刺激。通过半定量RT-PCR检查了表达量。结果，在肌腱愈合后切割和减少跟腱后，Keyaki的表达水平升高。

项目成果

Sakae Sano, Takahisa Sasho, Yuichi Wada, et al.: "Effects of Stretch on Gene Expression in Muscles and Tendon"J.Jpn.Orthop.Assoc.. 74. S1465 (2000)

Sakae Sano、Takahisa Sasho、Yuichi Wada 等：“拉伸对肌肉和肌腱基因表达的影响”J.Jpn.Orthop.Assoc.. 74.S1465 (2000)

Takahisa Sasho: "Cloning and Analysis of the Gene Induced by Cyclic Mechanical Stimuli"SIROT abst book. 38. (1999)

Takahisa Sasho：“循环机械刺激诱导的基因的克隆和分析”SIROT Abst 书。

Takahisa Sasho: "Cloning and analysis of the gene induced by cyclic mechanical stimuli"SIROT abstract book. 38 (1999)

Takahisa Sasho：《循环机械刺激诱导基因的克隆与分析》SIROT 摘要书。

Takahisa Susho: "Cloning and Analysis of the Gene Induced by Cyclic Mechanical Stimuli."SIROT abst book. 38. 38 (1999)

Takahisa Susho：“循环机械刺激诱导的基因的克隆和分析”。SIROT 摘要书。

佐野栄 他: "筋腱組織での伸張刺激の有無が遺伝子発現に与える影響"日本整形外科学会誌. 74. S1465 (2000)

Sakae Sano 等人：“肌肉肌腱组织中存在或不存在拉伸刺激对基因表达的影响”日本骨科协会杂志 74. S1465 (2000)。