Department of Neurobiology, Neuroscience Institute, Morehouse School of Medicine, Atlanta, GA 30310, USA.
Biochem Biophys Res Commun. 2011 Dec 16;416(3-4):337-42. doi: 10.1016/j.bbrc.2011.11.037. Epub 2011 Nov 15.
Circadian rhythm is a fundamental biological system involved in the regulation of various physiological functions. However, little is known about a nature or function of circadian clock in human primary cells. In the present study, we have applied in vitro real time circadian rhythm monitoring to study human clock properties using primary skin fibroblasts. Among factors that affect human physiology, slightly lower extracellular pH was chosen to test its effects on circadian rhythm expression. We established human primary fibroblast cultures obtained from three healthy subjects, stably delivered a circadian reporter gene Bmal1-luciferase, and recorded circadian rhythms in the culture medium at pH 7.2 and 6.7. At pH 7.2, robust and sustained circadian rhythms were observed with average period length 24.47 ± 0.03 h. Such rhythms were also found at pH 6.7; however, period length was significantly shortened to 22.60 ± 0.20, amplitude was increased, and damping rate was decreased. The effect of exposure to low pH on the period length was reversible. The shortened period was unlikely caused by factors affecting cell viability because cell morphology and MTT assay showed no significant difference between the two conditions. In summary, our results showed that the circadian rhythm expression is affected at pH 6.7 in human primary fibroblasts without affecting cell viability.
昼夜节律是调节各种生理功能的基本生物系统。然而，关于人类原代细胞中昼夜节律钟的性质或功能知之甚少。在本研究中，我们应用体外实时昼夜节律监测，使用原代皮肤成纤维细胞研究人类时钟特性。在影响人类生理的因素中，选择稍低的细胞外 pH 值来测试其对昼夜节律表达的影响。我们建立了来自 3 名健康受试者的人原代成纤维细胞培养物，稳定地传递昼夜节律报告基因 Bmal1-荧光素酶，并在 pH 7.2 和 6.7 的培养基中记录昼夜节律。在 pH 7.2 时，观察到强大而持续的昼夜节律，平均周期长度为 24.47 ± 0.03 h。在 pH 6.7 时也发现了这种节律；然而，周期长度明显缩短至 22.60 ± 0.20，振幅增加，衰减率降低。暴露于低 pH 值对周期长度的影响是可逆的。这种缩短的周期不太可能是由于影响细胞活力的因素造成的，因为细胞形态和 MTT 测定显示两种条件之间没有显著差异。总之，我们的结果表明，在不影响细胞活力的情况下，pH 6.7 会影响人原代成纤维细胞中昼夜节律的表达。
