A method for detecting reactive oxygen species and mitochondrial membrane potential in isolated perfused hearts

A technology of mitochondrial membrane potential and detection method, which can be used in material excitation analysis, fluorescence/phosphorescence, etc., and can solve problems such as continuous heart beating

Inactive Publication Date: 2017-02-22
喻秋珺
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Another problem that needs to be solved is that the heart keeps beating while maintaining normal perfusion during the imaging acquisition process. The experimental equipment and perfusion system also need to be improved accordingly to ensure the effective acquisition of images.

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  • A method for detecting reactive oxygen species and mitochondrial membrane potential in isolated perfused hearts
  • A method for detecting reactive oxygen species and mitochondrial membrane potential in isolated perfused hearts
  • A method for detecting reactive oxygen species and mitochondrial membrane potential in isolated perfused hearts

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Embodiment 1

[0026] A method for detecting reactive oxygen species and mitochondrial membrane potential in isolated perfused hearts, specifically a kind of O 2 - A real-time in situ detection method comprising the following steps:

[0027] The first step is the establishment of the isolated heart perfusion model. Pentobarbital sodium (50 mg / kg, i.p.) anesthetized and heparinized (500 IU / kg, i.v.) rabbits were quickly opened and the heart was fixed in the Langendorff perfusion system. Aortic retrograde perfusion with 95% O 2 +5%CO 2 Mixed gas saturated K-H solution (37°C, pH 7.4, mmol / L): NaCl 115, NaHCO 3 25, glucose11, KCl 4.0, CaCl 2 1.5, MgSO 4 0.9, KH 2 PO 4 0.9, in order to measure the systolic function in real time, cut an opening at the entrance of the pulmonary vein in the left atrium, insert a balloon connected to the pressure sensor into the left ventricle through the left atrium, completely discharge the air in the balloon and fill it with deionized water until it reaches...

Embodiment 2

[0036] A method for detecting reactive oxygen species and mitochondrial membrane potential in isolated perfused hearts, specifically a myocardial cell mitochondrial O 2 - A real-time in situ detection method comprising the following steps:

[0037] The first step, the establishment of rat isolated heart perfusion model, pentobarbital sodium (50mg / kg, i.p.) anesthetized and heparinized (500IU / kg, i.v.) rats, quickly open the chest to remove the heart and fix it in Langendorff perfusion system, transaortic retrograde perfusion with 95% O 2 +5%CO 2 Mixed gas saturated K-H solution (37°C, pH 7.4, mmol / L): NaCl 115, NaHCO 3 25, glucose 11, KCl 4.0, CaCl 2 1.5, MgSO 4 0.9, KH 2 PO 4 0.9, in order to measure the systolic function in real time, cut an opening at the entrance of the pulmonary vein in the left atrium, insert a balloon connected to the pressure sensor into the left ventricle through the left atrium, completely discharge the air in the balloon and fill it with deio...

Embodiment 3

[0046] A method for detecting reactive oxygen species and mitochondrial membrane potential in isolated perfused hearts, specifically a method for detecting H in cardiomyocytes during ischemia-reperfusion in isolated perfused hearts of mice 2 o 2 and a method for real-time in situ detection of mitochondrial membrane potential, comprising the following steps:

[0047]The first step is the establishment of isolated mouse heart perfusion model. Pentobarbital sodium (50mg / kg, i.p.) anesthetized and heparinized (500IU / kg, i.v.) mice were quickly removed from the chest and fixed in Langendorff perfusion system, transaortic retrograde perfusion with 95% O 2 +5%CO 2 Mixed gas saturated K-H solution (37°C, pH 7.4, mmol / L): NaCl 115, NaHCO 3 25, glucose 11, KCl 4.0, CaCl 2 1.5, MgSO 4 0.9, KH 2 PO 4 0.9, in order to measure the systolic function in real time, cut an opening at the entrance of the pulmonary vein in the left atrium, insert a balloon connected to the pressure sensor ...

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Abstract

The invention relates to a detection method for isolated perfusion heart active oxygen and mitochondrial membrane potential, which comprises the following steps of: establishing an isolated perfusion heart ischemia reperfusion model; realizing correlation of an isolated heart perfusion system, a probe loading device and a laser scanning confocal microscope; further carrying out real-time loading and microscope imaging on a fluorescence probe of the active oxygen and mitochondrial membrane potential; and finally, through image processing and data analysis, the method can be used for real-time and positioning measurement of heart ROS (reactive oxygen species) and mitochondrial membrane potential in various isolated heart models taking acute overload, ischemia reperfusion and the like as representatives under a stable perfusion state. The detection method has the characteristics of directness, accuracy, visibility, time and space positioning, and synchronized measurement of beating cardiac metabolism and function, and has the advantages of low cost, high efficiency, and good reliability and repeatability.

Description

technical field [0001] The invention relates to the technical field of detection of intracellular reactive oxygen species and mitochondrial membrane potential, in particular to a detection method for isolated perfused heart reactive oxygen species and mitochondrial membrane potential. [0002] technical background [0003] Reactive Oxygen Species (ROS) are derivatives of oxygen free radicals and non-free radical oxygen formed in the oxidation reaction of organisms, among which superoxide anion and hydrogen peroxide are the most common and most active. Oxidative stress induced by ROS is an important inducing factor for a series of cardiovascular diseases. Therefore, accurate detection of ROS content in cells and the mitochondrial membrane potential closely related to it is of great significance for the research and prevention of major cardiovascular diseases. [0004] Due to the extremely short lifetime and extremely low steady-state concentration of reactive oxygen species in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
Inventor 喻秋珺王海昌
Owner 喻秋珺
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