研究目的
Investigating the role of Src kinase in intracellular signaling pathways triggered by low-power infrared radiation in sensory neurons.
研究成果
Src kinase is involved in intracellular signaling pathways triggered by low-power IR radiation in sensory neurons, acting through the Na,K-ATPase/Src complex to modulate NaV1.8 channel voltage sensitivity and inhibit neurite growth. This provides a novel mechanism for signal transduction distinct from G proteins, with potential applications in pain relief.
研究不足
The study is limited to sensory neurons and may not generalize to other cell types. The patch-clamp method has inherent errors such as series resistance and run-down effect. The specific molecular interactions within the Na,K-ATPase/Src complex require further elucidation.
1:Experimental Design and Method Selection:
The study used patch-clamp method to investigate NaV
2:8 channel voltage sensitivity and organotypic nerve tissue culture to assess neurite growth. The Almers' limiting-slope procedure was applied to evaluate the effective charge (Zeff) of NaV8 channel activation gating system. Sample Selection and Data Sources:
Dissociated sensory neurons from dorsal root ganglia of newborn Wistar rats and explants from 10-12 days old chicken embryos were used.
3:List of Experimental Equipment and Materials:
CO2 laser (wavelength
4:6 μm), IMO-2N power meter, patch-clamp L/M-EPC 7 amplifier, Axio Observer Z1 microscope, semiconductor laser (wavelength 635 μm), optical wedges, mechanical screens, disk attenuator, mirrors, diaphragms, water filter, Petri dishes, collagen-coated dishes, СО2-incubator (Sanyo), reagents from Sigma. Experimental Procedures and Operational Workflow:
For patch-clamp, neurons were irradiated with low-power IR radiation (energy density 1*10^-11 J/cm2) with or without Src kinase inhibitor PP2 (10 μM). Ionic currents were recorded, and Zeff was calculated. For organotypic culture, explants were irradiated (energy density 2*10^-10 J/cm2) with or without PP2, and neurite growth was assessed by area index.
5:Data Analysis Methods:
Statistical analysis used one-way ANOVA followed by Tukey's test. Zeff was evaluated using the Almers' limiting-slope procedure. Image analysis used ImageJ and ZEN_2012 software.
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Microscope
Axio Observer Z1
Carl Zeiss
Visualizing explants and neurite growth in organotypic culture
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CO2 laser
Source of low-power infrared radiation for activating Na,K-ATPase transducer function in sensory neurons
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IMO-2N power meter
IMO-2N
Volgograd pilot plant
Monitoring radiation power in the optical setup
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Patch-clamp amplifier
L/M-EPC 7
Recording ionic currents from sensory neurons in whole-cell configuration
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Semiconductor laser
Visualizing the position of the CO2 laser beam
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CO2-incubator
Sanyo
Maintaining culture conditions for organotypic nerve tissue
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PP2
Sigma
Specific Src kinase inhibitor used to block IR radiation effects
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Collagenase
Type 1A
Sigma
Enzymatic treatment for dissociating sensory neurons
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Pronase E
Sigma
Enzymatic treatment for dissociating sensory neurons
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Tetrodotoxin
Sigma
Blocking fast tetrodotoxin-sensitive sodium channels to isolate NaV1.8 currents
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