Detection method for isolated perfusion heart active oxygen and mitochondrial membrane potential

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: 2012-07-25
喻秋珺
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 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.

Method used

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  • Detection method for isolated perfusion heart active oxygen and mitochondrial membrane potential
  • Detection method for isolated perfusion heart active oxygen and mitochondrial membrane potential
  • Detection method for isolated perfusion heart active oxygen and mitochondrial membrane potential

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Embodiment 1

[0025] A method for detecting reactive oxygen species and mitochondrial membrane potential of the isolated perfused heart, specifically a kind of O 2 - The real-time in-situ detection method includes the following steps:

[0026] The first step is to establish an isolated heart perfusion model. The rabbits are anesthetized with sodium pentobarbital (50mg / kg, ip) and heparinized (500IU / kg, iv). The heart is quickly opened to remove the heart and fixed in the Langendorff perfusion system. Aortic retrograde perfusion with 95% O 2 +5% CO 2 K-H liquid saturated with mixed gas (37℃, 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 of the heart in real time, cut an opening at the entrance of the left atrium and pulmonary vein, insert a balloon connected to the pressure sensor into the left ventricle through the left atrium, completely exhaust the air in the balloon and fill it with deionized water u...

Embodiment 2

[0035] A method for detecting reactive oxygen species and mitochondrial membrane potential of isolated perfused heart, specifically a myocardial cell mitochondria O during acute overload of calcium load in isolated rat perfused heart 2 - The real-time in-situ detection method includes the following steps:

[0036] In the first step, the rat isolated heart perfusion model was established. The rats were anesthetized with sodium pentobarbital (50mg / kg, ip) and heparinized (500IU / kg, iv), and the heart was quickly opened and fixed in Langendorff for perfusion. System, retrograde perfusion through the aorta with 95% O 2 +5% CO 2 K-H liquid saturated with mixed gas (37℃, 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 of the heart in real time, cut an opening at the entrance of the left atrium and pulmonary vein, insert a balloon connected to the pressure sensor into the left ventricle through th...

Embodiment 3

[0045] A method for detecting reactive oxygen species and mitochondrial membrane potential of isolated perfused heart, specifically a method for detecting H in myocardial cells during ischemia-reperfusion of isolated perfused heart of mice 2 O 2 And the real-time in-situ detection method of mitochondrial membrane potential includes the following steps:

[0046] In the first step, the mouse isolated heart perfusion model was established. The mice were anesthetized with sodium pentobarbital (50mg / kg, ip) and heparinized (500IU / kg, iv). The heart was quickly opened and fixed in Langendorff for perfusion. System, retrograde perfusion through the aorta with 95% O 2 +5% CO 2 K-H liquid saturated with mixed gas (37℃, 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 of the heart in real time, cut an opening at the entrance of the left atrium and pulmonary vein, insert a balloon connected to the pres...

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

Claims

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Application Information

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