Satellite cell number and cell cycle kinetics in response to acute myotrauma in humans: immunohistochemistry versus flow cytometry
Satellite cell number and cell cycle kinetics in response to acute myotrauma in humans: immunohistochemistry versus flow cytometry
复制标题
DOI：
10.1113/jphysiol.2010.190876
复制
发表时间：
2010-09-01
影响因子：
5.5
通讯作者：
Parise, Gianni
中科院分区：
文献类型：
--
作者：
McKay, Bryon R.;Toth, Kyle G.;Parise, Gianni
在人类中，肌肉卫星细胞计数是一种重要的测量方法，用于确定对各种刺激的肌源性反应。迄今为止，计数的标准做法是使用针对常见SC标记（Pax7， NCAM）的抗体的免疫组织化学（IHC）。流式细胞术（FC）分析可以更快速、更定量地确定SC池的变化，并有可能进行标准IHC难以实现的附加分析。在这项研究中，FC分析显示，运动引起的肌肉损伤（300次单侧最大偏心收缩）24小时后，从50毫克新鲜组织中分离的每毫克Pax7+细胞数量增加了36%。Pax7和神经细胞粘附分子（NCAM）的IHC分析似乎充分且相似地代表了损伤后sc的扩增（增加28-36%）。IHC和FC数据表明，Pax7是肌肉横截面中表达最广泛的SC标记物，代表了大多数阳性细胞，而NCAM的表达程度较低。此外，损伤后24小时，FC和IHC表现出相似的百分比变化（增加36%，Pax7；增加28%，NCAM）。对分离的sc进行FC分析显示，损伤24 h后细胞周期G(2)/M期每毫克Pax7+细胞数量增加202%。每毫克G(0)/G(1)的细胞数和s期细胞数分别增加32%和59%。在这里，我们说明使用FC作为一种方法，以每毫克组织为基础枚举SC数，提供更容易理解的肌肉质量关系（相对于肌核百分比或每肌纤维）。虽然IHC是一种强大的SC分析工具，但FC是一种快速、可靠和有效的SC定量方法，也是一种更有用的人类SC群体细胞周期动力学方法。
In humans, muscle satellite cell (SC) enumeration is an important measurement used to determine the myogenic response to various stimuli. To date, the standard practice for enumeration is immunohistochemistry (IHC) using antibodies against common SC markers (Pax7, NCAM). Flow cytometry (FC) analysis may provide a more rapid and quantitative determination of changes in the SC pool with potential for additional analysis not easily achievable with standard IHC. In this study, FC analysis revealed that the number of Pax7+ cells per milligram isolated from similar to 50 mg of fresh tissue increased 36% 24 h after exercise-induced muscle injury (300 unilateral maximal eccentric contractions). IHC analysis of Pax7 and neural cell adhesion molecule (NCAM) appeared to sufficiently and similarly represent the expansion of SCs after injury (28-36% increase). IHC and FC data illustrated that Pax7 was the most widely expressed SC marker in muscle cross-sections and represented the majority of positive cells, while NCAM was expressed to a lesser degree. Moreover, FC and IHC demonstrated a similar percentage change 24 h after injury (36% increase, Pax7; 28% increase, NCAM). FC analysis of isolated SCs revealed that the number of Pax7+ cells per milligram in G(2)/M phase of the cell cycle increased 202% 24 h after injury. Number of cells per milligram in G(0)/G(1) and cells in S-phase increased 32% and 59% respectively. Here we illustrate the use of FC as a method for enumerating SC number on a per milligram tissue basis, providing a more easily understandable relation to muscle mass (vs. percentage of myonuclei or per myofibre). Although IHC is a powerful tool for SC analysis, FC is a fast, reliable and effective method for SC quantification as well as a more informative method for cell cycle kinetics of the SC population in humans.