ROS-based method for detecting oxidative stress level of stichopus japonicus in breeding period
Through flow cytometry detection method, ROS is used to detect gonadal tissue and body cavity fluid during the reproductive period of ginseng, which solves the problem of low detection accuracy in the prior art, and achieves rapid and accurate detection of ginseng oxidative stress levels, ensuring health monitoring of ginseng reproduction and growth.
Patent Information
- Application Number
- CN202510624109.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-15
AI Technical Summary
In the prior art, methods for detecting the oxidative stress level of galactus ginseng have problems of low accuracy and low efficiency, especially in the detection of the galactus ginseng in the breeding period, it is difficult to accurately respond to the oxidative stress state of galactus.
The flow cytometry detection method was used to detect the ROS level in gonadal tissue or body cavity fluid during the reproduction of ginseng, as an indicator of oxidative stress, and the reactive oxygen was labeled with a DCFH-DA probe and tested in combination with a flow cytometer.
It realizes more accurate and faster detection of oxidative stress levels of galaxy ginseng, provides more reliable detection methods, ensures monitoring of galaxy reproduction and growth data, and fills the methodological gap in reproductive oxidative stress research.
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Figure CN120489667A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of aquatic animal physiology research, and in particular to a method for detecting the oxidative stress level of sea cucumbers during their reproductive period based on ROS. Background Art
[0002] Apostichopus japonicus, also known as sea cucumber, boasts immunity-boosting, blood-nourishing, and sperm-boosting properties, boasting high nutritional value and making it a valuable marine delicacy. With increasing demand for sea cucumber, the scale and production of sea cucumber aquaculture have continued to grow, fostering diverse aquaculture models, including shallow-sea seeding and proliferation, and factory-based intensive aquaculture. This has provided significant new impetus for increasing fishermen's income and the development of high-value fisheries.
[0003] Studies have found that sea cucumbers will produce oxidative stress when they encounter damage or environmental threats during their life history. During the reproduction process of sea cucumbers, due to the high energy metabolism, they will also cause oxidative stress in sea cucumbers, which will lead to aging and damage to the sea cucumber body. The physiological health of sea cucumbers is one of the important indicators for maintaining the reproduction and individual growth of sea cucumbers, and it is also the cornerstone of the sustainable development of the sea cucumber industry. Superoxide dismutase, catalase, reduced glutathione peroxidase, etc. are key markers of oxidative stress levels. Their activity levels are closely related to the degree of oxidative stress. Currently, researchers mostly use this type of oxidative stress-related enzymes to determine whether sea cucumbers have oxidative stress. However, this detection method has the disadvantages of the possibility of inaccurate reflection of protein expression differences and oxidation levels, delayed detection results, and low accuracy. Summary of the Invention
[0004] In order to solve the above technical problems, the present invention provides a method for detecting the oxidative stress level of sea cucumbers during the reproductive period based on ROS, so as to achieve the purpose of improving the detection efficiency and detection accuracy.
[0005] To achieve the above object, the technical solution of the present invention is as follows:
[0006] A ROS-based method for detecting oxidative stress levels in sea cucumbers during their reproductive period uses flow cytometry to detect ROS generated during the reproduction process of sea cucumbers as an indicator for detecting oxidative stress levels.
[0007] In the above scheme, ROS generated in the gonadal tissue or coelomic fluid during the reproduction of sea cucumbers are detected.
[0008] In a further technical solution, the detection method comprises the following steps:
[0009] 1) Obtain fresh gonadal tissue from sea cucumbers, soak it in 3×PBS, mince the tissue with sterilized scissors, place it in a glass homogenizer, add 3×PBS, and grind it to obtain a tissue suspension; alternatively, obtain fresh coelomic fluid from sea cucumbers, add 3×PBS, and mix thoroughly by pipetting to obtain a cell suspension;
[0010] 2) Filter through a 300-mesh silk sieve or cell sieve. After filtration, add 3× PBS and mix by pipetting repeatedly. Centrifuge the cell suspension at 1200 rpm for 6 minutes. Repeat 1-2 times, sieving the cell suspension each time.
[0011] 3) After centrifugation, remove the supernatant from the sample and add 3× PBS. Mix the lower layer of precipitated cells by pipetting to obtain a cell suspension. Pipette the cell suspension, filter it through a 300-mesh sieve or cell sieve, and add 3× PBS to prepare 1 ml of sea cucumber cell suspension.
[0012] 4) The ROS content was detected using a ROS detection kit and flow cytometry.
[0013] Preferably, in step 3), the concentration of the prepared 1 ml sea cucumber cell suspension is controlled at 1×10 6 ~1×10 7 .
[0014] In a further technical solution, the detection method of step 4) is as follows:
[0015] Add DCFH-DA to 1 ml of sea cucumber cell suspension and incubate at 37°C for 20 minutes or more, invert and mix every 3 to 5 minutes. Place the solution that has fully combined with the probe into a centrifuge at 1000 rpm / min for 5 minutes, aspirate the supernatant, and wash with PBS 1 to 2 times; resuspend the pellet after centrifugation in 3×PBS, place it in a flow cytometer, and set the channel to FITC fluorescence detection conditions for detection.
[0016] In a further technical solution, in step 1), the experiment is performed on the middle part of the reproductive tubules of the selective glandular tissue.
[0017] In a further technical solution, in step 1), the body cavity fluid needs to be pure body cavity fluid that has not been contaminated by damaged gonads or intestines during the dissection process.
[0018] Through the above technical solution, the present invention provides a method for detecting oxidative stress levels in sea cucumbers during their reproductive period based on ROS, which has the following beneficial effects:
[0019] During their reproduction, sea cucumbers produce reactive oxygen species (ROS), which cause oxidative stress in them, impacting parental health and future reproductive costs. This invention uses ROS directly as a detection indicator, enabling more accurate and rapid detection of oxidative stress levels in sea cucumbers. This provides a feasible and accessible detection method for future scientific research, thereby providing technical methods and references for cellular research at the tissue level in echinoderms.
[0020] Gonadal tissue, as the breeding tissue of sea cucumbers, can ensure the health of offspring. The coelomic fluid, as the main immune defense site of sea cucumbers, can reflect the health level of the sea cucumber body. Therefore, focusing on the gonadal tissue and coelomic fluid of sea cucumbers can provide guarantees for the healthy development of the sea cucumber industry.
[0021] This technical method provides innovative technical means for existing research, breaks through the technical bottleneck of ROS detection in sea cucumber germ cells, and establishes a standardized flow cytometry detection system for sea cucumber gonadal tissue for the first time. The detection efficiency and accuracy have been improved, filling the methodological gap in the study of sea cucumber reproductive oxidative stress to ensure the comprehensiveness of sea cucumber physiological research, and providing technical support for researchers to monitor sea cucumber reproduction and growth data. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.
[0023] Figure 1 Schematic diagram of flow cytometry results of ROS in gonadal cells of three female sea cucumbers; (a) is the fluorescein isothiocyanate level of female individual 1; (b) is the fluorescein isothiocyanate level of female individual 2; (c) is the fluorescein isothiocyanate level of female individual 3;
[0024] Figure 2 Schematic diagram of flow cytometry results of ROS in gonadal cells of three male sea cucumbers; (a) is the fluorescein isothiocyanate level of male individual 1; (b) is the fluorescein isothiocyanate level of male individual 2; (c) is the fluorescein isothiocyanate level of male individual 3;
[0025] Figure 3 Schematic diagram of flow cytometry results of ROS in coelomocytes of three female sea cucumbers; (a) is the fluorescein isothiocyanate level of female individual 1; (b) is the fluorescein isothiocyanate level of female individual 2; (c) is the fluorescein isothiocyanate level of female individual 3;
[0026] Figure 4Schematic diagram of flow cytometry results of ROS in coelomocytes of three male sea cucumbers; (a) is the fluorescein isothiocyanate level of male individual 1; (b) is the fluorescein isothiocyanate level of male individual 2; (c) is the fluorescein isothiocyanate level of male individual 3. DETAILED DESCRIPTION
[0027] The technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present invention.
[0028] The present invention provides a method for detecting the oxidative stress level of sea cucumbers during the reproductive period based on ROS, comprising the following steps:
[0029] Example 1
[0030] 1) Obtain approximately 1 g of fresh gonadal tissue from sea cucumbers, soak it in 3× PBS, mince the tissue with sterilized scissors, place it in a glass homogenizer, add approximately 1 ml of 3× PBS, and grind to obtain a tissue suspension;
[0031] 2) Filter the cell suspension through a 300-mesh sieve or cell sieve. After filtration, add 3× PBS and mix thoroughly by pipetting repeatedly. Centrifuge the cell suspension at 1200 rpm for 6 minutes. Repeat 1 to 2 times. Sieve the cell suspension after each resuspension.
[0032] 3) Remove the supernatant from the centrifuged sample obtained in 2), add 2 ml of 3× PBS, and mix the lower precipitated cells by pipetting to obtain a cell suspension. Pipette an appropriate amount of the cell suspension, filter through a 300-mesh sieve or cell sieve, and add an appropriate amount of 3× PBS to prepare 1 ml of sea cucumber gonad cell suspension.
[0033] 4) Select an appropriate kit and perform the assay according to the kit instructions. Add an appropriate concentration of DCFH-DA to the cell suspension obtained in 3) and incubate at 37°C for 20 minutes or longer, mixing by inversion every 3-5 minutes. Centrifuge the solution that has fully bound to the probe at 1000 rpm for 5 minutes, discard the supernatant, add 1 ml of 3× PBS staining buffer to resuspend the cells, centrifuge at 1000 rpm for 3-4 minutes, discard the supernatant, add 1 ml of 3× PBS to resuspend the cells, and centrifuge at 1000 rpm for 3-4 minutes.
[0034] 5) After centrifugation, the cell pellet obtained in 4) was resuspended in an appropriate amount of 3× PBS, placed in a flow cytometer, and detected using the FITC fluorescence detection channel.
[0035] Example 2
[0036] 1) Obtain approximately 1-2 ml of fresh coelomic fluid from sea cucumbers, add an equal proportion of 3× PBS, and mix thoroughly by pipetting to obtain a cell suspension.
[0037] 2) Filter the cell suspension through a 300-mesh sieve or cell sieve. After filtration, add 3× PBS and mix thoroughly by pipetting repeatedly. Centrifuge the cell suspension at 1200 rpm for 6 minutes. Repeat 1 to 2 times. Sieve the cell suspension after each resuspension.
[0038] 3) Remove the supernatant from the centrifuged sample obtained in 2), add 2 ml of 3× PBS, and mix the lower precipitated cells by pipetting to obtain a cell suspension. Pipette an appropriate amount of the cell suspension, filter through a 300-mesh sieve or cell sieve, and add an appropriate amount of 3× PBS to prepare 1 ml of sea cucumber gonad cell suspension.
[0039] 4) Select an appropriate kit and perform the assay according to the kit instructions. Add an appropriate concentration of DCFH-DA to the cell suspension obtained in 3) and incubate at 37°C for 20 minutes or longer, mixing by inversion every 3-5 minutes. Centrifuge the solution that has fully bound to the probe at 1000 rpm for 5 minutes, discard the supernatant, add 1 ml of 3× PBS staining buffer to resuspend the cells, centrifuge at 1000 rpm for 3-4 minutes, discard the supernatant, add 1 ml of 3× PBS to resuspend the cells, and centrifuge at 1000 rpm for 3-4 minutes.
[0040] 5) After centrifugation, the cell pellet obtained in 4) was resuspended in an appropriate amount of 3× PBS, placed in a flow cytometer, and detected using the FITC fluorescence detection channel.
[0041] Analysis of test results:
[0042] The test result of above-described embodiment 1 is as follows Figure 1 and Figure 2 As shown, from Figure 1 The average values of FITC in female gonadal cells in the three figures are 106892.33, 64897.14, and 102416.37, respectively. The average value of the three groups is 91401.95, which is comparable to the Figure 2 The average values of fluorescein isothiocyanate in the gonadal cells of three male individuals were: 97550.71, 61486.85, and 60144.28. The average of the three groups was 73060.61. Comparative analysis showed that the ROS level in females was higher than that in males. Therefore, the oxidative stress level in female gonadal tissue was higher than that in males.
[0043] The test results of above-described embodiment 2 are as follows Figure 3 and Figure 4 As shown, from Figure 3The average values of FITC in female coelomocytes are 154796.99, 125956.36, and 121774.40, respectively. The average value of the three groups is 134175.92, which is comparable to the Figure 4 The average values of fluorescein isothiocyanate in the coelomocytes of three male individuals were: 82462.27, 180961.09, and 129245.90. The average of the three groups was 130889.75. A comparative analysis found that the ROS level in females was higher than that in males, so the oxidative stress level in the coelomocytes of females was higher than that of males.
[0044] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for detecting oxidative stress levels in sea cucumbers during their reproductive period based on ROS, characterized in that: Flow cytometry was used to detect ROS produced during the reproduction of sea cucumbers as an indicator of oxidative stress level.
2. A method for detecting oxidative stress levels in sea cucumbers during their reproductive period based on ROS according to claim 1, wherein Detection of ROS produced in gonadal tissue or coelomic fluid during sea cucumber reproduction.
3. a kind of sea cucumber breeding period oxidative stress level detection method based on ROS according to claim 1 or 2, is characterized in that, The steps include: 1) Obtain fresh gonadal tissue from sea cucumbers, soak it in 3×PBS, mince the tissue with sterilized scissors, place it in a glass homogenizer, add 3×PBS, and grind it to obtain a tissue suspension; alternatively, obtain fresh coelomic fluid from sea cucumbers, add 3×PBS, and mix thoroughly by pipetting to obtain a cell suspension; 2) Filter through a 300-mesh silk sieve or cell sieve. After filtration, add 3× PBS and mix by pipetting repeatedly. Centrifuge the cell suspension at 1200 rpm for 6 minutes. Repeat 1-2 times, sieving the cell suspension each time. 3) After centrifugation, remove the supernatant from the sample and add 3× PBS. Mix the lower layer of precipitated cells by pipetting to obtain a cell suspension. Pipette the cell suspension, filter it through a 300-mesh sieve or cell sieve, and add 3× PBS to prepare 1 ml of sea cucumber cell suspension. 4) The ROS content was detected using a ROS detection kit and flow cytometry.
4. A method for detecting oxidative stress levels in sea cucumbers during their reproductive period based on ROS according to claim 3, wherein In step 3), the concentration of the prepared 1 ml sea cucumber cell suspension was controlled at 1×10 6 ~1×10 7 .
5. a kind of sea cucumber breeding period oxidative stress level detection method based on ROS according to claim 3, is characterized in that, The detection method of step 4) is as follows: Add DCFH-DA to 1 ml of sea cucumber cell suspension and incubate at 37°C for 20 minutes or more, invert and mix every 3 to 5 minutes. Place the solution that has fully combined with the probe into a centrifuge at 1000 rpm / min for 5 minutes, aspirate the supernatant, and wash with PBS 1 to 2 times; resuspend the pellet after centrifugation in 3×PBS, place it in a flow cytometer, and set the channel to FITC fluorescence detection conditions for detection.
6. A method for detecting oxidative stress levels in sea cucumbers during their reproductive period based on ROS according to claim 3, wherein In step 1), the experiment was performed on the middle part of the reproductive tubules of the selective glandular tissue.
7. A method for detecting oxidative stress levels in sea cucumbers during their reproductive period based on ROS according to claim 3, wherein In step 1), the body cavity fluid should be pure body cavity fluid that is not contaminated by damaged gonads or intestines during the dissection process.