Wozniak Ashley C, Pilipowicz Orest, Yablonka-Reuveni Zipora, Greenway Steven, Craven Shauna, Scott Elliott, Anderson Judy E
Department of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba R3E 0W3, Canada.
J Histochem Cytochem. 2003 Nov;51(11):1437-45. doi: 10.1177/002215540305101104.
Single-fiber cultures can be used to model satellite cell activation in vivo. Although technical deficiencies previously prevented study of stretch-induced events, here we describe a method developed to study satellite cell gene expression by in situ hybridization (ISH) using protocol modifications for fiber adhesion and fixation. The hypothesis that mechanical stretching activates satellite cells was tested. Fiber cultures were established from normal flexor digitorum brevis muscles and plated on FlexCell dishes with a layer of Vitrogen. After 2 hr of stretch in the presence of BrdU, satellite cells on fibers attached to Vitrogen were activated above control levels. In the absence of activating treatments or mechanical stretch, ISH studies showed 0-6 c-Met+ satellite cells per fiber. Time course experiments demonstrated stable quiescence in the absence of stretch and significant peaks in activation after 30 min and 2 hr of stretch. Frequency distributions for unstretched fiber cultures showed a significantly greater number of quiescent c-Met+ satellite cells than were activated by stretching, suggesting that typical activation stimuli did not trigger cycling in the entire c-Met+ population of satellite cells. These methods have a strong potential to further dissect the nature of stretch-induced activation and gene expression among characterized populations of individual quiescent and activated satellite cells.
单纤维培养可用于模拟体内卫星细胞的激活。尽管技术缺陷先前阻碍了对拉伸诱导事件的研究，但在此我们描述了一种通过原位杂交（ISH）研究卫星细胞基因表达的方法，该方法对纤维黏附与固定的方案进行了改进。对机械拉伸激活卫星细胞这一假说进行了验证。从正常的趾短屈肌建立纤维培养物，并接种于涂有一层维特罗（Vitrogen）的FlexCell培养皿中。在存在溴脱氧尿苷（BrdU）的情况下进行2小时拉伸后，附着于维特罗的纤维上的卫星细胞被激活至高于对照水平。在没有激活处理或机械拉伸的情况下，ISH研究显示每根纤维有0 - 6个c-Met +卫星细胞。时间进程实验表明，在无拉伸时卫星细胞处于稳定的静止状态，而在拉伸30分钟和2小时后激活出现显著峰值。未拉伸的纤维培养物的频率分布显示，静止的c-Met +卫星细胞数量显著多于因拉伸而被激活的细胞，这表明典型的激活刺激并未在整个c-Met +卫星细胞群体中触发细胞周期。这些方法极有可能进一步剖析单个静止和激活卫星细胞特征群体中拉伸诱导激活和基因表达的本质。
