Method for evaluating oxidative damage of dendritic cells
By analyzing the changes in specific metabolites in dendritic cells, the problem that the prior art cannot effectively detect slight oxidative damage in cells is solved, and a rapid, safe and effective assessment of oxidative damage in dendritic cells is achieved.
Patent Information
- Application Number
- CN202411826430.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-12
- Publication Date
- 2025-04-01
AI Technical Summary
The prior art methods for evaluating oxidative damage of dendritic cells are complex and time-consuming, and the reagents used are highly toxic, which are harmful to the health of the experimenter, and it is impossible to detect the slight oxidative damage state when the cells are not lethal.
By analyzing the changes in cell metabolites, the significant increase in metabolites such as glycine, cytosine, 3'-adenylate, dephosphorylation CoA, acetyl-CoA and porcine deoxygenated bile salt were detected by high-performance liquid chromatography-triple quadrupole mass spectrometer, as a biomarker for evaluating oxidative damage in dendritic cells.
The oxidative damage state of cells is realized without lethality, which reduces the experimental time and cost, and avoids the health risks of using harmful reagents to the experimenter.
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Figure CN119738509A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for evaluating oxidative damage of dendritic cells, belonging to the field of biotechnology. Background Art
[0002] Oxidative damage refers to the imbalance of the oxidative and antioxidant systems caused by an increase in free radicals in the body or a weakening of the body's antioxidant protection ability under the stimulation of harmful factors inside and outside the body. In this state, excessive accumulation of reactive oxygen species (ROS) can cause damage to biological molecules such as nucleic acids, proteins, and lipids, leading to the occurrence and development of certain chronic degenerative diseases. Antioxidants can achieve the purpose of antioxidant by neutralizing free radicals, scavenging oxidants, or preventing oxidants from converting into more toxic compounds, thereby slowing down or inhibiting cell damage. Therefore, studying the mechanism of oxidative damage caused by harmful factors and evaluating and screening effective antioxidants are of great significance for maintaining the health of the body and preventing and controlling diseases. There are many types of oxidative damage sources, among which hydrogen peroxide (H 2 O 2 ) is the most common, which can inhibit cell proliferation, cause oxidative damage to intracellular macromolecules, and ultimately lead to serious consequences such as cell aging, death and mutation. 2 O 2 Induced oxidative damage cell models are commonly used to explore the mechanisms of free radical-mediated cell damage and the protection and repair mechanisms of antioxidants against oxidative damage.
[0003] Dendritic cells are professional antigen-presenting cells in the immune system. They are the key link between innate immunity and adaptive immunity, and play a vital role in promoting immune defense and maintaining immune tolerance. Dendritic cells play an important role in sensing tissue damage and initiating tissue repair, but they are exposed to high levels of ROS during infiltration into inflammatory tissues. Existing studies have shown that after oxidative damage to tissues or cells, autophagy is dysregulated and cell apoptosis increases, leading to the release of cellular contents, which in turn causes changes in the metabolic response of dendritic cells.
[0004] At present, the screening for evaluating the oxidative damage of dendritic cells is mainly carried out by studying the toxic effect of samples on dendritic cells, using IC 50 The MTT method is the main indicator. The commonly used method is the MTT method, but the MTT method is complicated and time-consuming, and has high requirements for the operation of the experimenter. In addition, MTT is also carcinogenic, which is harmful to the health of the experimenter. Moreover, when the cells are not yet lethal, slight oxidative damage has occurred, and the MTT method used in the prior art cannot detect the state of the oxidative damage, resulting in missed detection. Therefore, it is particularly important to seek a more stable, efficient, environmentally friendly method for evaluating the oxidative damage activity of dendritic cells that can detect the non-lethal oxidative damage state of cells. Summary of the invention
[0005] [Technical issues]
[0006] The current screening methods for evaluating the oxidative damage of dendritic cells are complex and time-consuming, and the reagents used are highly toxic, which is harmful to the health of the experimenters. In addition, the MTT method commonly used to evaluate oxidative damage currently evaluates the occurrence of oxidative damage by the cell mortality rate, and lacks an evaluation method for cells that are only slightly oxidatively damaged but not lethal.
[0007] [Technical solution]
[0008] In order to overcome the shortcomings of the current prior art, the purpose of the present invention is to provide a method for evaluating dendritic cell oxidative damage based on cell metabolites as biomarkers, which is original, fast and efficient, and can reduce the time cost of evaluating dendritic cell oxidative damage methods. Metabolites are the basis of the phenotype of organisms. By analyzing the difference in metabolites between the experimental group and the control group or studying the biological processes in which the differential metabolites participate, it is helpful to reveal the mechanism of life activities in which they participate and complete research on regulatory pathways and other aspects. However, so far, no relevant reports on a method for evaluating oxidative damage based on dendritic cell metabolites as biomarkers have been found.
[0009] The present invention provides a method for evaluating cell oxidative damage, which evaluates the degree of cell oxidative damage according to changes in cell metabolites.
[0010] In one embodiment of the present invention, the cell metabolites include one or more of glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA, and hyodeoxycholate.
[0011] In one embodiment of the present invention, the method comprises the following steps:
[0012] S1. Cell sample processing: culture sample cells and control cells;
[0013] S2. Cell sample extraction: Take out the cell sample, slowly thaw it, add pre-cooled extractant or use liquid nitrogen for direct extraction, place it at room temperature for 10 minutes, add 1-3mL 80% methanol to extract intracellular metabolites. Collect the supernatant by centrifugation and filter it through a 0.22μm organic filter membrane;
[0014] S3. The supernatant obtained in step S2 is detected by high performance liquid chromatography-triple quadrupole mass spectrometry to analyze changes in cell metabolites.
[0015] In one embodiment of the present invention, the centrifugal treatment temperature in S2 is 4°C, the centrifugal speed is 6000-12000×g, and the centrifugal treatment time is 10-15 min.
[0016] In one embodiment of the present invention, the high performance liquid chromatography-triple quadrupole mass spectrometer condition parameters described in S3 are: Xbridge amide column for separation; column temperature 40°C; flow rate 0.35mL / min; injection volume 1μL; mobile phase composition A: water + 20mM ammonium acetate + 5% acetonitrile (ammonia water adjusted to pH 9), B: acetonitrile; gradient elution program is as follows: start-85% B, 0.01min, 85% B, 3.0min, 30% B, 12.0min, 2% B, 15.0min, 2% B, 16.0min, 85% B, 23.0min, 85% B. Electrospray ionization (ESI) positive ion and negative ion modes were used for detection respectively. MetaboAnalyst platform (https: / / www.metaboanalyst.ca / ) was used for data analysis.
[0017] In one embodiment of the present invention, the control cells in step S1 are cells that have not been damaged by oxidation.
[0018] In one embodiment of the present invention, in step S3, when one or more of the cell metabolites in the cell sample are significantly increased compared with the control cell supernatant, it indicates that the cells have suffered oxidative damage.
[0019] In one embodiment of the present invention, the cells include but are not limited to dendritic cells.
[0020] The present invention also provides a biomarker or a composition thereof for evaluating cell oxidative damage, wherein the biomarker or the composition thereof comprises one or more of glycine, cytosine, 3'-adenosine, dephospho-CoA, acetyl-CoA, and porcine deoxycholate, and the cells include but are not limited to dendritic cells.
[0021] The present invention also provides the use of glycine, cytosine, 3'-adenylic acid, dephosphorylated coenzyme A, acetyl coenzyme A and porcine deoxycholate in evaluating cell oxidative damage.
[0022] In one embodiment of the present invention, the oxidative damage of cells is evaluated based on the expression of glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA and hyodeoxycholate in the cells.
[0023] The present invention also provides the use of the method, or the biomarker or a combination thereof in screening functional products for alleviating oxidative damage to dendritic cells.
[0024] Beneficial effects:
[0025] The method for evaluating oxidative damage of dendritic cells of the present invention can screen out the oxidative damage state of cells when the cells have not yet died after oxidative damage; whereas in the prior art, oxidative damage is judged by cell mortality rate, and cells with slight oxidative damage cannot be screened out.
[0026] The method of the present invention can realize high-throughput screening, can reduce the time cost of evaluating the method of oxidative damage to dendritic cells, and can also reduce the time cost of searching for functional products that slow down oxidative damage to dendritic cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 A bar graph showing the screening of hydrogen peroxide concentration in Example 1 of the present invention;
[0028] Figure 2 The bar graph is a graph showing the expression difference folds of the significantly different metabolites in Example 1 of the present invention;
[0029] Figure 3 This is a bar graph of the expression difference folds of the significantly different metabolites in Example 2 of the present invention;
[0030] Figure 4 This is a bar chart of the multiples of metabolites that are not correlated with oxidative damage in Comparative Example 1 of the present invention. DETAILED DESCRIPTION
[0031] The embodiments of the present invention are described in detail below with reference to the examples. If no specific conditions are specified in the examples, the reaction is carried out according to conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.
[0032] The dendritic cells involved in the following embodiments are mouse dendritic cells DC 2.4 from Shanghai Meiyan Biotechnology Co., Ltd., the carrageenan is analytically pure κ-carrageenan from Shanghai Aladdin Reagent Co., Ltd., and the carrageenan oligosaccharides are prepared by a laboratory preliminary method. The specific structural characterization of carrageenan oligosaccharides has been published in the article ("Characterization and Gel Properties of Low-Molecular-Weight Carrageenans Prepared by Photocatalytic Degradation").
[0033] In order to verify the feasibility and reliability of the method provided by the present invention for evaluating oxidative damage of dendritic cells based on cell metabolites, Examples 1 and 2 are listed. The specific experimental methods and test results are as follows:
[0034] Example 1
[0035] H 2 O2 Treatment concentration screening: In this embodiment, H 2 O 2 In the concentration screening experiment of the oxidative damage model, a total of 9 concentration gradients were set, namely 0mM, 0.2mM, 0.4mM, 0.6mM, 1.0mM, 1.3mM, 1.6mM, 1.9mM and 2.4mM. Figure 1 For different H 2 O 2 The cell survival rate bar graph under the conditions of concentration. 2 O 2 After H 2 O 2 When the concentration is greater than 0.6 mM, the 2 O 2 There were significant differences between the groups, H 2 O 2 When the concentration was 1.3 mM, the cell survival rate was 58.9%. 2 O 2 The oxidative damage model concentration was 1.3 mM for subsequent experiments.
[0036] In this embodiment, a control group and H 2 O 2 Treatment group, in which the control group (denoted by Ctrl) contained DMEM medium and dendritic cells were not treated; H 2 O 2 Treatment group (with H 2 O 2 (indicated) containing DMEM medium, dendritic cells and a final concentration of 1.3 mM H 2 O 2 to be processed.
[0037] Using H 2 O 2 The dendritic cells are processed, the cell metabolites are statistically analyzed, and the oxidative damage of the dendritic cells is evaluated, including the following steps:
[0038] S1. Take the dendritic cells in the logarithmic growth phase, count them, and adjust the concentration of the mixed solution to 1.0×10 per well. 5 Inoculate cells at a density of 100 cells / mL into a 12-well plate, add 1 mL of DMEM medium, culture at 37°C for 24 h, and discard the supernatant. 2 O 2 DMEM medium (H 2 O 2 The cells were incubated at 37°C for 24 h in DMEM medium (treated group) and DMEM medium (control group), respectively, and the supernatant was discarded. The cells were then washed three times with sterile PBS.
[0039] The cultured cells can be directly processed, or frozen and stored for a period of time before processing. The cryopreservation method is: discard the supernatant after centrifugation, quickly freeze the collected cells with pre-cooled PBS buffer, and then store at low temperature.
[0040] S2. Take out the cell sample (if it is a frozen sample, it needs to be thawed slowly), add pre-cooled extractant or use liquid nitrogen for direct extraction, leave it at room temperature for 10 minutes, add 1mL 80% methanol to extract intracellular metabolites. Collect the supernatant by centrifugation, pass it through a 0.22μm organic filter membrane, and test it on the machine.
[0041] S3. The supernatant obtained in step S2 is detected by high performance liquid chromatography-triple quadrupole mass spectrometry to analyze changes in cell metabolites.
[0042] S4. The differential metabolites of dendritic cells after sample treatment (glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA, porcine deoxycholate) were used as biomarkers to evaluate the oxidative damage of dendritic cells based on their differential metabolism.
[0043] The experimental data used the increase of six metabolites, namely glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA, and porcine deoxycholate, as the basis for judging the oxidative damage of dendritic cells and evaluating the oxidative damage of dendritic cells. Figure 2 This is a bar graph of the expression differences of the significant differences in metabolites in this example, where H 2 O 2 In the treated group, glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA, and hyodeoxycholate increased significantly, suggesting that the addition of H 2 O 2 After treatment, dendritic cells will suffer from oxidative damage. This method can be used to evaluate the ability of dendritic cells to sustain oxidative damage, and the method has good reliability.
[0044] Example 2
[0045] In this embodiment, H is set 2 O 2 treatment group, carrageenan treatment group and carrageenan oligosaccharide treatment group, among which H 2 O 2 Treatment group (with H 2 O 2 (indicated) containing DMEM medium, dendritic cells and a final concentration of 1.3 mM H 2 O 2 Carrageenan treatment group (with H 2 O 2+KC indicates) containing DMEM medium, dendritic cells in the presence of 1.3 mM H 2 O 2 Carrageenan oligosaccharide treatment group (with H 2 O 2 +KCO) containing DMEM medium, dendritic cells were added with 1.3 mM H 2 O 2 Then carrageenan oligosaccharide was added for treatment.
[0046] Dendritic cells were treated with carrageenan and carrageenan oligosaccharides, and cell metabolites were statistically analyzed to evaluate oxidative damage of dendritic cells, including the following steps:
[0047] S1. Take the dendritic cells in the logarithmic growth phase, count them, and adjust the concentration of the mixed solution to 1.0×10 per well. 5 Inoculate cells into 6-well plates at a density of 100 cells / mL, add 2 mL of DMEM medium, culture at 37°C for 24 h, and discard the supernatant. 2 O 2 The cells were incubated at 37°C for another 24 h, the supernatant was discarded, and the cells were washed 5 times with sterile PBS. 2 O 2 +KC group) or carrageenan oligosaccharide (H 2 O 2 +KCO group) DMEM solution 2mL, or DMEM medium (H 2 O 2 The cells of the control group (Ctrl group) were cultured with 2 mL of culture medium at 37°C for 24 h; the supernatant was aspirated and the cells were washed 5 times with sterile PBS. The cells of the control group (Ctrl group) were cultured with reference to the above method, except that hydrogen peroxide, carrageenan, and carrageenan oligosaccharide were not added, and only DMEM medium was used.
[0048] The cultured cells can be directly processed, or frozen and stored for a period of time before processing. The cryopreservation method is: discard the supernatant after centrifugation, quickly freeze the collected cells with pre-cooled PBS buffer, and then store at low temperature.
[0049] S2. Take out the cell sample (if it is a frozen sample, it needs to be thawed slowly), add pre-cooled extractant or use liquid nitrogen for direct extraction, leave it at room temperature for 10 minutes, add 1mL 80% methanol to extract intracellular metabolites. Collect the supernatant by centrifugation, pass it through a 0.22μm organic filter membrane, and test it on the machine.
[0050] S3. The supernatant obtained in step S2 is detected by high performance liquid chromatography-triple quadrupole mass spectrometry to analyze changes in cell metabolites.
[0051] S4. The differential metabolites of dendritic cells after sample treatment (glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA, porcine deoxycholate) were used as biomarkers to evaluate the oxidative damage of dendritic cells based on their differential metabolism.
[0052] The experimental data used the increase of six metabolites, namely glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA, and porcine deoxycholate, as the basis for judging the oxidative damage of dendritic cells and evaluating the oxidative damage of dendritic cells. Figure 3 This is a bar graph of the expression differences of the significant differences in metabolites in this example, where H 2 O 2 In the treatment group, glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA, and hyodeoxycholate increased significantly, while after treatment with carrageenan or carrageenan oligosaccharides that alleviate oxidative damage, glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA, and hyodeoxycholate decreased significantly, which verified that the addition of H 2 O 2 After treatment, oxidative damage will occur in dendritic cells. Glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA, and porcine deoxycholate can be used as marker metabolites for evaluating oxidative damage in dendritic cells. This method has good reliability.
[0053] Comparative Example 1:
[0054] Specific implementation method is as in Example 1, except that the metabolites of carbamoyl phosphate, α-ketoglutaric acid and phenylpropiolic acid are detected, and the results are as follows: The method described in this example does not cause changes in the three compounds in dendritic cells ( Figure 4 ), suggesting that these three compounds cannot be used as a basis for judging oxidative damage of dendritic cells.
[0055] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. A method for evaluating cell oxidative damage, characterized in that: The degree of oxidative damage of cells is evaluated according to the changes in cell metabolites, wherein the cell metabolites include one or more of glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA, and hyodeoxycholate.
2. The method according to claim 1, characterized in that: The method comprises the following steps: (1) Cultivating sample cells and control cells; (2) after cultivation, collecting the cell supernatant and detecting the content of the metabolite; (3) Comparing the changes in the content of the cell metabolites in the supernatant of the cell sample and the control cells.
3. The method according to claim 2, characterized in that: The control cells in step (1) are cells that have not been damaged by oxidation.
4. The method according to claim 3, characterized in that: In step (3), when one or more of the cell metabolites in the cell sample is significantly increased compared to the control cell supernatant, it indicates that the cells have suffered oxidative damage.
5. The method according to claim 4, characterized in that: The cells include, but are not limited to, dendritic cells.
6. A biomarker for evaluating cellular oxidative damage or a composition thereof, characterized in that: The biomarker or the combination thereof includes one or more of glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA, and hyodeoxycholate.
7. The biomarker or the composition thereof according to claim 6, characterized in that: The cells include, but are not limited to, dendritic cells.
8. Application of glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA and porcine deoxycholate in evaluating cellular oxidative damage.
9. The use according to claim 8, characterized in that: The oxidative damage of cells was evaluated based on the expression of glycine, cytosine, 3'-adenylic acid, dephospho-CoA, acetyl-CoA and porcine deoxycholate in the cells.
10. Use of the method according to any one of claims 1 to 5, or the biomarker according to claim 6 or 7 or a combination thereof in screening functional products that mitigate oxidative damage to dendritic cells.