Method for dynamically monitoring release of nitric oxide by endothelial cells in real time by using total internal reflection fluorescence microscopic imaging system

Abstract

The invention discloses a method for dynamically monitoring release of nitric oxide by endothelial cells in real time by using a total internal reflection fluorescence microscopic imaging system. The method comprises the following steps: (1) separating vascular endothelial cells and identifying the vascular endothelial cells; (2) adding endothelial cells into a cell culture dish for adherent culture; (3) incubating the endothelial cells with the DAF-FM in a dark place; (4) carrying out a fluorescence detection experiment; (5) NIS-Elements AR software on the TIRFM-EMCCD machine system is opened, and main parameters are set well; (6) recording the change of the fluorescence value of the cells, starting to add Ach after the fluorescence value tends to be stable, and observing the dynamic reaction of the cells to Ach according to the change of the fluorescence value; and (7) after the experiment is finished, analyzing experimental data and drawing. According to the invention, DAF-FM and TIRFM are combined for the first time, and an experimental method capable of dynamically detecting the NO synthesis capability of living cells in real time and relatively quantifying the NO synthesis capability is found.

Description

[0001] This application is a divisional application of a patent application entitled "A method for real-time dynamic monitoring of nitric oxide release by endothelial cells using a total internal reflection fluorescence microscopy imaging system". The application date of the original application is March 8, 2021, and the application number is 202110251463.5. Technical Field

[0002] The invention relates to the field of biotechnology, in particular to a method for real-time dynamic monitoring of nitric oxide release by endothelial cells using a total internal reflection fluorescence microscopy imaging system. Background Art

[0003] Nitric oxide (NO) is an important messenger and effector molecule in the body. It was selected as a star molecule by Science magazine in 1992. Current research shows that NO and NO synthase (NOS) not only have a wide range of physiological functions in the cardiovascular, immune, and nervous systems, but also participate in the occurrence and development of many diseases. Since 1987, when studies confirmed that vascular endothelial cells can synthesize and release NO, the role of NO in the cardiovascular system has received much attention. American scientist Robert Fouchgott and others won the 1998 Nobel Prize in Physiology or Medicine for discovering that "NO acts as a signaling molecule in the cardiovascular system." It can be seen that the capture and dynamic monitoring of NO in vascular endothelial cells is of great significance to the study of the application of NO in organisms.

[0004] Vascular endothelial cells are continuous single-layer flat cells covering the surface of the vascular lumen. They can secrete a variety of vascular function regulatory factors and participate in the normal regulation of the body's vascular system. NO is the body's most important vasodilator substance, and NO synthesis usually reflects the state of vascular endothelial function. The mechanism of NO's action in improving cardiovascular and cerebrovascular functions: NO gas produced in vascular endothelial cells, because it is fat-soluble, quickly penetrates the cell membrane and diffuses downward into smooth muscle cells, thereby acting on smooth muscle cells to relax them, dilate blood vessels, and ultimately lead to a drop in blood pressure. At the same time, it will also quickly penetrate the cell membrane and diffuse upward into the blood, enter platelet cells, reduce platelet activity, inhibit their aggregation and adhesion to the vascular endothelium, thereby preventing the formation of thrombi and the occurrence of atherosclerosis.

[0005] NO determination includes direct method and indirect method, among which direct method mainly includes: diazotization method, chemiluminescence method, gas chromatography and mass spectrometry. The most commonly used methods for indirect determination of NO are nitrite colorimetry and quantitative determination of NOS. DAF-FM, namely 3-Amino, 4-aminomethyl-2', 7'-difluorescein, diacetate, is the latest generation of fluorescent probes for quantitative detection of NO. DAF-FM can pass through the cell membrane and can be catalyzed by esterases in the cell to form DAF-FM that cannot pass through the cell membrane after entering the cell. DAF-FM can produce strong fluorescence after reacting with NO, with an excitation wavelength of 495nm and an emission wavelength of 515nm. Any instrument that can detect fluorescein, including fluorescence microscope, flow cytometer, fluorescence spectrophotometer or fluorescence microplate reader, can be used for the detection of this fluorescent probe. None of these methods can dynamically monitor the dynamic reaction of endothelial cells synthesizing NO in real time.

[0006] NO was first discovered in 1935 by Humphrey Davy in his research on laughing gas (NO 2 O) was discovered by the American pharmacologist Robert F. Fouchgott, and was always considered a toxic gas molecule until 1980, when he published a paper in the famous journal Nature, pointing out that the vasodilating effect of acetylcholine (Ach) depends on NO released by the vascular endothelium. Since then, the academic community generally believes that NO is, and the biological and medical research of NO has developed rapidly. Acetylcholine is a neurotransmitter that can activate the endothelium to produce NO, thereby inducing endothelium-dependent vasodilation. It is usually used to detect the ability of the vascular endothelium to produce NO. Summary of the invention

[0007] In order to solve the problems existing in the above prior art, the object of the present invention is to provide a method for real-time dynamic monitoring of nitric oxide release from endothelial cells using a total internal reflection fluorescence microscopy imaging system, wherein the total internal reflection fluorescence microscopy imaging system is a total internal reflection fluorescence microscope electron-multiplying charge-coupled device (TIRFM-EMCCD) imaging system.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] 1. A method for real-time dynamic monitoring of nitric oxide release from endothelial cells using a total internal reflection fluorescence microscopy imaging system, characterized in that it comprises the following steps:

[0010] (1) Isolation of primary vascular endothelial cells in vitro using type 2 collagenase method, and identification of the isolated vascular endothelial cells using endothelial cell-specific marker CD31;

[0011] (2) After endothelial cells have been cultured for 2 generations, they are digested and resuspended with trypsin, and the cells are added to a cell culture dish at a density of 1000 cells per square millimeter and cultured for 2 hours;

[0012] (3) DAF-FM was diluted 1000 times with phosphate buffer saline (PBS) to a final concentration of 5 μM; after the cells were allowed to stand, the cell culture medium was removed and the diluted DAF-FM was added in a volume that could completely cover the bottom of the cell dish; the cells were placed in a 37°C cell culture incubator and incubated in the dark for 30 minutes; the cells were washed three times with phosphate buffer saline (PBS) at pH 7.4 to fully remove the DAF-FM that had not entered the cells. The entire process needed to be in the dark;

[0013] (4) Add 190 μL PSS solution into the culture dish for fluorescence detection experiment;

[0014] (5) Open the NIS-ElementsAR software on the TIRFM-EMCCD machine system and set the main parameters;

[0015] (6) After selecting a suitable cell field of view under the microscope, start recording the changes in cell fluorescence values, observe the fluorescence values ​​of the cells through the real-time dynamic curve graph of the fluorescence values, and start adding ACh after the fluorescence values ​​tend to be stable, and observe the dynamic response of the cells to ACh through the changes in the fluorescence values;

[0016] (7) After the experiment, the experimental data will be analyzed and graphed;

[0017] The PSS liquid formula is as follows: Nacl 137mM, Kcl 6mM, Mgcl 21mM, Cacl 22mM, Glucose 5mM, HEPES 10mM, pH adjusted to 7.4 with NaOH;

[0018] In step (5), the main parameters are set as follows: Zyla Settings parameter settings: Format: NoBinning, Auto Exposure: 100ms, ReadoutMode: Rolling shutter, Readout Rate: 540MHz, Dynamic Range: 12-bit&Gain4, SensorMode: Overlap; Ti Pad main parameter settings: Intensilight: 1, Filters: Green;

[0019] In the step (7), after the experiment, the experimental data was opened and analyzed using NIS-ElementsARAnalysis software, and the data was organized and plotted using GraphPad Prism 5.0 software.

[0020] Beneficial effects: The present invention provides a method for real-time dynamic monitoring of nitric oxide release from endothelial cells using a total internal reflection fluorescence microscopy imaging system. The present invention combines DAF-FM and TIRFM for the first time, and discovers an experimental method that can dynamically detect the NO synthesis ability of living cells in real time and can perform relative quantification. Total internal reflection fluorescence microscopy is used to study biological phenomena occurring on the surface of objects, where a series of important basic life reactions in cell and molecular biology occur. Compared with traditional fluorescence microscopy, it has a high signal-to-noise ratio, detects single-molecule fluorescence signals, and can rapidly perform real-time dynamic imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0022] Figure 1 The present invention is a real-time dynamic monitoring diagram of NO synthesis in vascular endothelial cells using the method of the present invention. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The present invention provides a method for real-time dynamic monitoring of nitric oxide release by endothelial cells using a total internal reflection fluorescence microscopy imaging system, comprising the following steps:

[0025] (1) Isolation of primary vascular endothelial cells in vitro using type 2 collagenase method, and identification of the isolated vascular endothelial cells using endothelial cell-specific marker CD31;

[0026] (2) After endothelial cells have been cultured for 2 generations, they are digested and resuspended with trypsin, and the cells are added to a cell culture dish at a density of 1000 cells per square millimeter and cultured for 2 hours;

[0027] (3) DAF-FM was diluted 1000 times with phosphate buffer saline (PBS) to a final concentration of 5 μM; after the cells were allowed to stand, the cell culture medium was removed and the diluted DAF-FM was added in a volume that could completely cover the bottom of the cell dish; the cells were placed in a 37°C cell culture incubator and incubated in the dark for 30 minutes; the cells were washed three times with phosphate buffer saline (PBS) at pH 7.4 to fully remove the DAF-FM that had not entered the cells. The entire process needed to be in the dark;

[0028] (4) Add 190 μL PSS solution to the culture dish for fluorescence detection experiment; wherein the PSS liquid formula is as follows: Nacl 137 mM, Kcl 6 mM, Mgcl 21 mM, Cacl 22 mM, Glucose 5 mM, HEPES 10 mM, pH adjusted to 7.4 with NaOH;

[0029] (5) Open the NIS-ElementsAR software on the TIRFM-EMCCD machine system and set the main parameters; the main parameter settings are as follows: Zyla Settings parameter settings: Format: No Binning, Auto Exposure: 100ms, Readout Mode: Rolling shutter, Readout Rate: 540MHz, Dynamic Range: 12-bit&Gain4, Sensor Mode: Overlap; Ti Pad main parameter settings: Intensilight: 1, Filters: Green;

[0030] (6) After selecting a suitable cell field of view under the microscope, start recording the changes in cell fluorescence values, observe the fluorescence values ​​of the cells through the real-time dynamic curve graph of the fluorescence values, and start adding ACh after the fluorescence values ​​tend to be stable, and observe the dynamic response of the cells to ACh through the changes in the fluorescence values;

[0031] (7) After the experiment, the experimental data were opened and analyzed using NIS-ElementsARAnalysis software, and the data were organized and graphed using GraphPad Prism 5.0 software.

[0032] like Figure 1 As shown in the figure, endothelial cells were stained with DAF-FM and showed green fluorescence. After selecting representative cells (circled in white), the TIRFM imaging system was used to monitor the dynamic changes of NO production in living cells. When Ach (acetylcholine) was added to the cell culture medium, the fluorescence intensity was significantly enhanced ( Figure 1 A), Figure 1A is a real-time dynamic change diagram of fluorescence intensity before and after adding different concentrations of Ach. The fluorescence value before adding Ach can locate F0, and the maximum fluorescence peak after adding Ach is Fmax. The ratio of F0 to Fmax can be used to relatively quantify the ability of endothelial cells to respond to Ach and the ability to synthesize NO. In addition, according to Figure 1 The X-axis (time axis) of C can obtain the time interval from F0-Fmax and Fmax-F1, so as to calculate the NO generation and degradation efficiency ( Figure 1 B and 1C).

[0033] In summary, the present invention combines DAF-FM and TIRFM for the first time and finds an experimental method that can dynamically detect the NO synthesis ability of living cells in real time and can perform relative quantification thereof.

[0034] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0035] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for real-time dynamic monitoring of nitric oxide release from endothelial cells using a total internal reflection fluorescence microscopy imaging system, characterized in that: The following steps are involved: (1) Isolation of primary vascular endothelial cells in vitro using type 2 collagenase method, and identification of the isolated vascular endothelial cells using endothelial cell-specific marker CD31; (2) After endothelial cells have been cultured for 2 generations, they are digested and resuspended with trypsin, and the cells are added to a cell culture dish at a density of 1000 cells per square millimeter and cultured for 2 hours; (3) DAF-FM was diluted 1000 times with phosphate buffer saline (PBS) to a final concentration of 5 μM; after the cells were allowed to stand, the cell culture medium was removed and the diluted DAF-FM was added in a volume that could completely cover the bottom of the cell dish; the cells were placed in a 37°C cell culture incubator and incubated in the dark for 30 minutes; the cells were washed three times with phosphate buffer saline (PBS) at pH 7.4 to fully remove the DAF-FM that had not entered the cells. The entire process needed to be in the dark; (4) Add 190 μL PSS solution into the culture dish for fluorescence detection experiment; (5) Open the NIS-ElementsAR software on the TIRFM-EMCCD machine system and set the main parameters; (6) After selecting a suitable cell field of view under the microscope, start recording the changes in cell fluorescence values, observe the fluorescence values ​​of the cells through the real-time dynamic curve graph of the fluorescence values, and start adding ACh after the fluorescence values ​​tend to be stable, and observe the dynamic response of the cells to ACh through the changes in the fluorescence values; (7) After the experiment, the experimental data will be analyzed and graphed; The PSS liquid formula is as follows: Nacl 137mM, Kcl 6mM, Mgcl 21mM, Cacl 22mM, Glucose 5mM, HEPES 10mM, pH adjusted to 7.4 with NaOH; In the step (5), the main parameter settings are as follows: Zyla Settings parameter settings: Format: NoBinning, Auto Exposure: 100ms, ReadoutMode: Rolling shutter, Readout Rate: 540MHz, DynamicRange: 12-bit&Gain4, SensorMode: Overlap; Ti Pad main parameter settings: Intensilight: 1, Filters: Green; In the step (7), after the experiment is completed, the experimental data is opened and analyzed using NIS-Elements ARAnalysis software, and the data is organized and graphed using GraphPadPrism 5.0 software.

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