Method for detecting oxidative stress level of stichopus japonicus in breeding period based on mitochondrial membrane potential
Through flow cytometry detection method, the mitochondrial membrane potential in the gonad tissue or body cavity fluid of the ginseng ginseng was detected using the JC-1 detection kit and flow cytometer, which solved the problem of ineffective evaluation of oxidative stress in the existing technology, achieved accurate monitoring of the ginseng ginseng oxidative stress level, and improved the research and industrial development of the ginseng reproduction.
Patent Information
- Application Number
- CN202510624137.2
- 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
The prior art has failed to effectively detect the oxidative stress level of prickly ginseng, especially the inhibitory effect on mitochondrial membrane potential during the breeding period, resulting in the inability to accurately evaluate ATP production and cellular function maintenance.
The flow cytometry detection method was used to detect the mitochondrial membrane potential in the gonadal tissue or body cavity fluid, and the mitochondrial membrane potential changes were analyzed using the JC-1 detection kit and flow cytometer to evaluate the level of oxidative stress.
The intuitive and rapid detection of the oxidative stress level of ginseng is achieved, the research level of ginseng breeding period has been improved, the quality and economic benefits of seedlings have been ensured, and the healthy development of the ginseng industry has been promoted.
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Figure CN120489668A_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 mitochondrial membrane potential. 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] Research has found that sea cucumbers experience oxidative stress during their life cycle when exposed to injury or environmental threats. The high energy metabolism during reproduction also contributes to oxidative stress in sea cucumbers, leading to aging and damage. Numerous studies have shown that oxidative stress can reduce mitochondrial membrane potential, thereby inhibiting adenosine triphosphate (ATP) production. Mitochondria are the primary site of ATP production in animal cells and are crucial organelles for cellular energy conversion and apoptosis. Mitochondria maintain oxidative phosphorylation through a membrane potential gradient generated by the electron transport chain, thereby driving ATP synthesis. During respiratory oxidation, mitochondria store the energy generated as electrochemical potential energy in the inner mitochondrial membrane, creating an asymmetric distribution of proton and other ion concentrations across the inner membrane and forming the mitochondrial membrane potential (MMP, ΔΨm). Normal MMP is a prerequisite for maintaining mitochondrial oxidative phosphorylation and ATP production, and stable MMP is crucial for maintaining normal cellular function. Therefore, studying mitochondrial membrane potential is crucial for monitoring oxidative stress levels in sea cucumbers. In the current research on oxidative stress in sea cucumbers, no reasonable method has been used to detect whether ATP production is inhibited after oxidative damage, nor has mitochondrial activation been used to determine whether the decrease in ATP content is due to mitochondrial inhibition. 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 mitochondrial membrane potential, so as to achieve the purpose of intuitively and effectively detecting the mitochondrial membrane potential and the subsequent effects caused by oxidative stress in the sea cucumber gonad tissue or body cavity fluid.
[0005] To achieve the above object, the technical solution of the present invention is as follows:
[0006] A method for detecting the oxidative stress level of sea cucumbers during their reproductive period based on mitochondrial membrane potential uses flow cytometry to detect the mitochondrial membrane potential formed during the reproduction process of sea cucumbers as an indicator for detecting the oxidative stress level.
[0007] In the above scheme, the mitochondrial membrane potential formed in the gonadal tissue or coelomic fluid during the reproduction of sea cucumbers is 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) Mitochondrial membrane potential was detected using JC-1 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 JC-1 staining working solution to 1 ml of sea cucumber cell suspension, mix by inversion several times, incubate at 37°C for 20 min or more, centrifuge at 600 rpm / min for 3-4 min, discard the supernatant, add 1 ml of 1×JC-1 staining buffer to resuspend the cells, centrifuge at 600 rpm / min for 3-4 min, discard the supernatant, add 1 ml of 1×JC-1 staining buffer to resuspend the cells, centrifuge at 600 rpm / min for 3-4 min, discard the supernatant, add 1 ml of 1×JC-1 staining buffer to resuspend the cells, centrifuge at 600 rpm / min for 3-4 min; resuspend the cell pellet after centrifugation with 1×JC-1 staining buffer, 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 method for detecting the oxidative stress level of sea cucumbers during the reproductive period based on mitochondrial membrane potential provided by the present invention has the following beneficial effects:
[0019] 1. The present invention adopts a method for detecting the oxidative stress level of sea cucumbers during the reproductive period based on mitochondrial membrane potential, which can intuitively and effectively detect the mitochondrial membrane potential and oxidative stress level in the sea cucumber gonad tissue or body cavity fluid.
[0020] 2. The present invention can significantly improve the research level of the sea cucumber breeding period, promote the innovation and development of the sea cucumber breeding industry, and improve the quality of sea cucumber seedlings and future economic benefits.
[0021] The mitochondrial membrane potential level studied in the present invention is the main indicator of reduced energy production in sea cucumbers due to oxidative stress. By detecting the mitochondrial membrane potential level in the sea cucumber gonads or body cavity fluid, the body state and gamete quality of sea cucumber parents under different breeding or environmental conditions can be effectively monitored, the quality of sea cucumber seedlings and the hatching rate of offspring can be guaranteed, and the health level of offspring can be effectively improved. This method has a short detection time and can make up for the shortcomings of incomplete and non-specific scientific research data. It can be widely used in sea cucumber parent breeding research. 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 JC-1 gonadal cells of female sea cucumbers; (a) is the forward scattered light intensity / side scattered light intensity graph; (b) is the phycoerythrin / side angle scattered light intensity graph; (c) is the fluorescein isothiocyanate content graph;
[0024] Figure 2 Schematic diagram of flow cytometry results of male sea cucumber gonadal tissue cells JC-1; (a) is the forward scattered light intensity / side scattered light intensity graph; (b) is the phycoerythrin / side angle scattered light intensity graph; (c) is the fluorescein isothiocyanate content graph;
[0025] Figure 3 Schematic diagram of flow cytometry results of female sea cucumber coelomocyte JC-1; (a) is the forward scattered light intensity / side scattered light intensity graph; (b) is the phycoerythrin / side angle scattered light intensity graph; (c) is the fluorescein isothiocyanate content graph;
[0026] Figure 4 Schematic diagram of flow cytometry results of male sea cucumber coelomocyte JC-1; (a) is the forward scattered light intensity / side scattered light intensity graph; (b) is the phycoerythrin / side angle scattered light intensity graph; (c) is the fluorescein isothiocyanate content graph;
[0027] Note: Forward scattered light intensity / side scattered light intensity: cell size / cell granularity; phycoerythrin / side scattered light intensity. In the figure: B is the percentage of JC-1 aggregates; FITC amount: JC-1 aggregate amount. DETAILED DESCRIPTION
[0028] 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.
[0029] The present invention provides a method for detecting the oxidative stress level of sea cucumbers during the reproductive period based on mitochondrial membrane potential, which is as follows:
[0030] Example 1
[0031] 1) Obtain approximately 1 g of fresh gonadal tissue from sea cucumbers, soak it in 3xPBS, mince the tissue with sterilized scissors, place it in a glass homogenizer, add approximately 1 ml of 3xPBS, and grind to obtain a cell suspension.
[0032] 2) Filter through a 300-mesh sieve or cell sieve. After filtration, add 3× PBS reagent and repeatedly pipette to mix. Centrifuge the cell suspension at 1200 rpm for 6 minutes. Repeat 1 to 2 times. Sieve the cell suspension after each resuspension.
[0033] 3) Remove the supernatant from the centrifuged sample obtained in 2), add 2 ml of 3× PBS, and mix the lower layer of 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 gonadal or coelomocyte suspension.
[0034] 4) Select a JC-1 detection kit and perform the test according to the kit instructions. Add an appropriate concentration of JC-1 staining working solution to the cell suspension obtained in 3) and mix by inversion several times. Incubate at 37°C for 20 minutes or more. Centrifuge at 600 rpm / min for 3-4 minutes, discard the supernatant, add 1 ml of 1× JC-1 staining buffer to resuspend the cells, centrifuge at 600 rpm / min for 3-4 minutes, discard the supernatant, add 1 ml of 1× JC-1 staining buffer to resuspend the cells, and centrifuge at 600 rpm / min for 3-4 minutes.
[0035] 5) Resuspend the cell pellet after centrifugation in 4) with an appropriate amount of 1×JC-1 staining buffer, place it in a flow cytometer, and set the channel to FITC fluorescence detection conditions for detection.
[0036] Example 2
[0037] 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.
[0038] 2) Filter through a 300-mesh sieve or cell sieve. After filtration, add 3× PBS reagent and repeatedly pipette to mix. Centrifuge the cell suspension at 1200 rpm for 6 minutes. Repeat 1 to 2 times. Sieve the cell suspension after each resuspension.
[0039] 3) Remove the supernatant from the centrifuged sample obtained in 2), add 2 ml of 3× PBS, and mix the lower layer of 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 gonadal or coelomocyte suspension.
[0040] 4) Select a JC-1 detection kit and perform the test according to the kit instructions. Add an appropriate concentration of JC-1 staining working solution to the cell suspension obtained in 3) and mix by inversion several times. Incubate at 37°C for 20 minutes or more. Centrifuge at 600 rpm / min for 3-4 minutes, discard the supernatant, add 1 ml of 1× JC-1 staining buffer to resuspend the cells, centrifuge at 600 rpm / min for 3-4 minutes, discard the supernatant, add 1 ml of 1× JC-1 staining buffer to resuspend the cells, and centrifuge at 600 rpm / min for 3-4 minutes.
[0041] 5) Resuspend the cell pellet after centrifugation in 4) with an appropriate amount of 1×JC-1 staining buffer, place it in a flow cytometer, and set the channel to FITC fluorescence detection conditions for detection.
[0042] Analysis of test results:
[0043] The test result of above-described embodiment 1 is as follows Figure 1 and Figure 2 As shown, from Figure 1 (a) shows that the cell granularity of the female gonadal tissue is Figure 2 (a) The cell granularity in the male gonadal tissue is relatively large. Figure 1 (b) shows that the JC-1 aggregates activated by cells in the gonadal tissue of female individuals are 0.03%. Figure 2 (b) The JC-1 aggregates in the male gonad tissue activated by cells were 0.03%, Figure 1(c) It can be seen that the number of unactivated JC-1 cells in the gonadal tissue of female individuals is lower than Figure 2 (c) The number of unactivated JC-1 in the male gonads. In summary, the mitochondrial activity in the gonadal tissue of male individuals is higher than that of female individuals.
[0044] The test results of above-described embodiment 2 are as follows Figure 3 and Figure 4 As shown, from Figure 3 (a) shows that the cell granularity ratio of the female gonadal tissue is Figure 4 (a) The cell granularity in the male gonadal tissue is relatively small. Figure 3 (b) shows that the JC-1 aggregates activated by cells in the gonadal tissue of female individuals are 0.0%. Figure 4 (b) The JC-1 aggregates in the male gonad tissue activated by cells were 1.07%, Figure 3 (c) It can be seen that the number of unactivated JC-1 cells in the gonadal tissue of female individuals is higher than that in Figure 4 (c) The number of unactivated JC-1 in the male gonads. In summary, the mitochondrial activity in the gonadal tissue of male individuals is higher than that of female individuals.
[0045] 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 mitochondrial membrane potential, characterized in that: Flow cytometry was used to detect the mitochondrial membrane potential formed 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 reproductive period based on mitochondrial membrane potential according to claim 1, characterized in that, Detection of mitochondrial membrane potential formed 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 mitochondrial membrane potential according to claim 1, 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) Mitochondrial membrane potential was detected using JC-1 detection kit and flow cytometry.
4. a kind of sea cucumber breeding period oxidative stress level detection method based on mitochondrial membrane potential according to claim 3, is characterized in that, 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 mitochondrial membrane potential according to claim 3, is characterized in that, The detection method of step 4) is as follows: Add JC-1 staining working solution to 1 ml of sea cucumber cell suspension, mix by inversion several times, incubate at 37°C for 20 min or more, centrifuge at 600 rpm / min for 3-4 min, discard the supernatant, add 1 ml of 1×JC-1 staining buffer to resuspend the cells, centrifuge at 600 rpm / min for 3-4 min, discard the supernatant, add 1 ml of 1×JC-1 staining buffer to resuspend the cells, centrifuge at 600 rpm / min for 3-4 min, discard the supernatant, add 1 ml of 1×JC-1 staining buffer to resuspend the cells, centrifuge at 600 rpm / min for 3-4 min; resuspend the cell pellet after centrifugation with 1×JC-1 staining buffer, 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 mitochondrial membrane potential 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 mitochondrial membrane potential 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.