CLONING OF MECHANOSENSITIVE RECEPTORS

细胞在化学、光、机械刺激和细胞外应激下存活。机械应力的生物感知包括听觉、平衡、触觉、肌肉和肌腱运动的检测以及体积扩张的注意。机械刺激包括摩擦、压力和拉伸。细胞通过细胞内代谢来适应这些刺激。响应机械应力的离子通道活性的变化是清楚的。然而，离子通道本身是否是机械敏感受体尚不清楚。它们没有被发现的一个原因是它们很小，在细胞中很稀少。尽管如此，最近分子生物学研究技术的发展使解决这一问题成为可能。我们的研究采用了以下几种策略：1)基于聚合酶链反应的方法克隆机械敏感受体cdna。然而，我们鉴定了已经报道过的cdna；2)培养细胞正压装置的研制。这台仪器正在进行研究；凝胶内蛋白激酶测定法和mRNA差异显示法被用于研究细胞内第二信使系统的变化，并发现响应细胞外正压的新基因。

CELL IS ALIVE UNDER CHEMICAL,LIGHT AND MECHANICAL STIMULI AND EXTRACELLULAR STRESS.BIOLOGICAL PERCEPTION OF MECHANICAL STRESS INCLUDES HEARING,BALANCE,TOUCH SENSATION,DETECTION OF MOVEMENT OF MUSCLE AND TENDON,AND NOTICE OF VOLUME EXPANSION.MECHANICAL STIMULI INCLUDE FRICTION,PRESSURE AND STRETCH.CELLS ADAPT TO THESE STIMULI BY INTRACELLULAR METABOLISM.CHANGE IN ACTIVITY OF ION CHANNELS RESPONDING TO MECHANICAL STRESS IS CLEAR.HOWEVER,IT IS NOT CLEAR THAT ION CHANNELS ARE MECHANOSENSITIVE RECEPTORS THEMSELVES OR NOT.ONE REASON WHY THEY ARE NOT FOUND IS THAT THEY ARE TINY AND SCARCE IN CELLS.IN SPITE OF THIS SITUATION RECENT DEVELOPMENT OF RESEARCH TECHNIQUES IN MOLECULAR BIOLOGY MAKES IT POSSIBLE TO SOLVE THE PROBLEM.OUR STUDY APPLIED SEVERAL STRATE GIES AS FOLLOWS : 1) CLONING OF MECHANOSENSITIVE RECEPTOR cDNAs BY POLYMERASE CHAIN REACTION BASED METHODS.HOWEVER,WE IDENTIFIED cDNAs ALREADY REPORTED ; 2) DEVELOPMENT OF APPARATUS OF POSITIVE PRESSURE ON CULTURE CELLS.THIS APPARATUS IS UNDER USE TO ONGOING STUDY ; 3) IN-GEL PROTEIN KINASE ASSAY METHOD AND 4) mRNA DIFFERENTIAL DISPLAY METHOD ARE UNDER USE TO STUDY CHANGE IN INTRACELLULAR SECOND MESSENGER SYSTEMS AND TO FIND NOVEL GENES WHICH RESPOND TO EXTRACELLULAR POSITIVE PRESSURE.