Takanashi Y, Weir B K, Vollrath B, Kasuya H, Macdonald R L, Cook D
Division of Neurosurgery, University of Alberta, Edmonton, Canada.
Neurosurgery. 1992 Mar;30(3):346-50. doi: 10.1227/00006123-199203000-00006.
A culture of smooth muscle cells obtained from monkey middle cerebral arteries was developed to allow quantitative assessment of intracellular calcium and immunofluorescence analysis after various periods of exposure to oxyhemoglobin. Intracellular calcium concentration was examined for up to 7 days after a single exposure to oxyhemoglobin. Intracellular calcium concentrations were measured with the fluorescent dye fura-2 and were significantly elevated for 7 days after exposure to oxyhemoglobin (P less than 0.01). Less than 2 minutes after application of oxyhemoglobin, there was marked elevation of intracellular calcium from the control value of 75 +/- 2 nmol/L to 240 +/- 28 nmol/L (P less than 0.01 by analysis of variance). Intracellular calcium concentration of cells exposed for 24 hours to oxyhemoglobin and then grown in normal oxyhemoglobin-free medium fell close to normal levels on Days 3 and 7. On Day 3, the increase in intracellular calcium that followed repeated daily exposure to oxyhemoglobin was greater than that resulting from a single application of oxyhemoglobin (P less than 0.01 by Student's t test), but by Day 7 the elevation produced by these different approaches was similar. Smooth muscle cells exposed to oxyhemoglobin showed a reduction in immunoreactivity to alpha-actin. These data support the hypothesis that disruption of intracellular calcium regulation and calcium overloading may be important in the process of cell injury, which results in vasoconstriction and sometimes cell death, after exposure to oxyhemoglobin.
我们培养了从猴大脑中动脉获取的平滑肌细胞，以便在暴露于氧合血红蛋白不同时间段后，对细胞内钙进行定量评估并进行免疫荧光分析。单次暴露于氧合血红蛋白后，对细胞内钙浓度进行长达7天的检测。用荧光染料fura - 2测量细胞内钙浓度，暴露于氧合血红蛋白后7天内，钙浓度显著升高（P小于0.01）。施加氧合血红蛋白后不到2分钟，细胞内钙从对照值75±2 nmol/L显著升高至240±28 nmol/L（方差分析，P小于0.01）。暴露于氧合血红蛋白24小时后再在无正常氧合血红蛋白的培养基中培养的细胞，其细胞内钙浓度在第3天和第7天接近正常水平。在第3天，每日重复暴露于氧合血红蛋白后细胞内钙的增加大于单次施加氧合血红蛋白所致的增加（Student t检验，P小于0.01），但到第7天，这些不同方法产生的升高相似。暴露于氧合血红蛋白的平滑肌细胞对α - 肌动蛋白的免疫反应性降低。这些数据支持以下假说：细胞内钙调节紊乱和钙超载在暴露于氧合血红蛋白后导致细胞损伤的过程中可能起重要作用，细胞损伤会导致血管收缩，有时还会导致细胞死亡。
