An Fengwei, Xing Lijun, Zhang Zhiqiang, Chen Lei
Department of Otorhinolaryngology, Jinan Military General Hospital, Jinan, 250031, Shandong Province, China.
Eur Arch Otorhinolaryngol. 2015 Oct;272(10):2839-45. doi: 10.1007/s00405-014-3354-2. Epub 2014 Nov 4.
We aimed to investigate the effect of histamine on ciliary beat frequency (CBF) through combining high-speed digital microscopy and patch-clamp technology. Ciliated cells were obtained from septum and turbinate of 90-120-day-old healthy male guinea pigs. Tight seal was formed by applying negative pressure on the glass electrode after the drawing and pushing progress. Then, we enrolled high-speed digital microscopy to measure CBF before and after treatment with histamine of different concentrations ranging from 10(-6) to 10(-1) mol/L in Hank's solution and D-Hank's solution as well as after administrating adenosine triphosphate. One-way ANOVA, Student's t test or Kruskal-Wallis test was used for statistical comparisons. Glass electrode fix up ciliated cell is available at tip diameter of 2-5 μm and negative pressure of 10-20 cmH2O column. The baseline CBF in Hank's solution was higher than in D-Hank's solution. Treatment with 10(-6)-l0(-3) mol/L histamine of concentrations can stimulate a rise of CBF. Nevertheless, CBF in all groups decreased to baseline CBF within 20 min. Generally, 10(-2) mol/L histamine can stimulate a rise of CBF; meanwhile, the high concentration of histamine killed 50% ciliated cell. Histamine at 10(-1) mol/L killed all ciliated cells. Ciliary beating activity decreased in Ca(2+)-free solution. Moreover, adenosine triphosphate could increase CBF effectively after the stimulation effect of histamine. We construct an effective technology integrating patch-clamp technique with CBF measurements on ciliated cells. Extracellular histamine stimulation could increase CBF effectively.
我们旨在通过结合高速数字显微镜和膜片钳技术来研究组胺对纤毛摆动频率(CBF)的影响。从90 - 120日龄健康雄性豚鼠的鼻中隔和鼻甲获取纤毛细胞。在拉制和推压过程后,通过对玻璃电极施加负压形成紧密封接。然后,我们采用高速数字显微镜测量在汉克溶液和D - 汉克溶液中不同浓度(范围从10⁻⁶至10⁻¹mol/L)组胺处理前后以及给予三磷酸腺苷后的CBF。采用单因素方差分析、学生t检验或克鲁斯卡尔 - 沃利斯检验进行统计学比较。玻璃电极固定纤毛细胞时,尖端直径为2 - 5μm,负压为10 - 20 cmH₂O柱。汉克溶液中的基线CBF高于D - 汉克溶液中的。10⁻⁶ - 10⁻³mol/L浓度的组胺处理可刺激CBF升高。然而,所有组的CBF在20分钟内降至基线CBF。一般来说,10⁻²mol/L组胺可刺激CBF升高;同时,高浓度组胺使50%的纤毛细胞死亡。10⁻¹mol/L组胺使所有纤毛细胞死亡。在无钙溶液中纤毛摆动活性降低。此外,在组胺的刺激作用后,三磷酸腺苷可有效增加CBF。我们构建了一种将膜片钳技术与纤毛细胞CBF测量相结合的有效技术。细胞外组胺刺激可有效增加CBF。