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目的:建立组织块法体外培养嗅觉受体神经元(ORNs)细胞内游离钙离子浓度([Ca2+]i)的实时观察系统,分析嗅觉信号转导体系中几个关键环节的作用。方法:以双激发波长钙离子荧光探针Fura-2 AM孵育培养的ORNs,通过双波长比率法,实时定量观察ORNs的[Ca2+]i。分别以Forskolin和IBMX刺激ORNs,观察[Ca2+]i的改变。分别耗竭细胞内钙库和使用不含钙离子的细胞外液,判断细胞内[Ca2+]i的来源。结果:静息ORNs的[Ca2+]i为(58.5±12.8)nmol/L。Forskolin和IBMX刺激可使ORNs的[Ca2+]i快速升高,且作用可逆。二者所引发的钙信号来自于细胞外钙内流,而非细胞内钙库的释放。结论:本研究成功建立了实时定量观察ORNs细胞内[Ca2+]i的系统,ORNs的[Ca2+]i受第二信使门控钙离子通道的调节。
OBJECTIVE: To establish a real-time observation system of intracellular free calcium concentration ([Ca2 +] i) in olfactory receptor neurons (ORNs) cultured in vitro by tissue block method and to analyze the role of several key links in olfactory signal transduction system. Methods: The cultured ORNs were incubated with Fura-2 AM, a dual-excitation calcium ion probe, and the [Ca2 +] i of ORNs was quantitatively observed by dual-wavelength ratio method. ORS were stimulated with Forskolin and IBMX respectively to observe the changes of [Ca2 +] i. Respectively depleted intracellular calcium stores and the use of extracellular free calcium ions to determine the source of intracellular [Ca2 +] i. Results: The [Ca2 +] i of resting ORNs was (58.5 ± 12.8) nmol / L. Forskolin and IBMX stimulation can cause ORNs [Ca2 +] i rapid increase, and the role of reversible. The calcium signals elicited by both are derived from the influx of extracellular calcium rather than the release of intracellular calcium stores. Conclusion: This study successfully established a real-time quantitative observation of ORNs intracellular [Ca2] i system, ORNs [Ca2] i by the second messenger gated calcium channel regulation.