Method for inducing autophagy of crab blood cells by rapamycin

Abstract

The invention belongs to the technical field of cell biological detection, and particularly relates to a method for inducing crab blood cell autophagy by rapamycin, which comprises the following steps: wiping the base parts of the last pair of swimming feet of a crab body by using an alcohol cotton ball; injecting the rapamycin working solution into the crab body; hemolymph of crabs is collected and prepared into blood cell suspension; and preparing the blood cell suspension into a blood cell detection solution, and carrying out autophagy level detection. The effect of inducing blood cell autophagy by rapamycin is confirmed by detecting autophagy level, lysosome activity and active oxygen relative content, and detection results of a laser confocal microscope and a flow cytometer are used for mutual verification, so that a new reference is provided for improving crab blood cell functions and improving breeding effect, and the method has a wide application prospect. The sustainable development of the shrimp and crab culture industry is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of cell biology detection, and particularly relates to a method for rapamycin to induce crab blood cell autophagy. Background Art

[0002] Hemocytes are an important defense mechanism for crustaceans to resist hypoxia, low temperature, ammonia nitrogen and heavy metal stress, and play an important role in maintaining normal life activities of organisms. Autophagy is a metabolic process inside eukaryotic cells, which can provide energy and maintain the stability of the internal environment of cells by breaking down damaged macromolecules and organelles in cells. In crabs, autophagy may also be involved in regulating the ammonia nitrogen metabolism process. Some studies have shown that autophagy can affect the metabolism and utilization of ammonia nitrogen by crabs. When the environmental ammonia nitrogen concentration is high, crabs may respond to ammonia nitrogen stress by increasing autophagy activity.

[0003] Rapamycin is an immunosuppressant and anti-tumor drug. It has also been found to regulate the cellular autophagy process by inhibiting the mTOR signaling pathway. In recent years, the study of autophagy has become a hot topic, and rapamycin has also become a focus. Although rapamycin-induced blood cell autophagy has been widely studied in mammals and other model organisms, there are relatively few studies on the autophagy function of crabs, and most of the work is based on frozen samples. The autophagy research method based on isolated cells has not been systematic in crabs, which limits the in vivo functional analysis, so there are few related reports. Summary of the invention

[0004] The purpose of the present invention is to provide a method for inducing crab blood cell autophagy with rapamycin, thereby overcoming the shortcomings of the prior art, providing a practical basis for improving crab blood cell function by exploring the induction of crab autophagy by rapamycin, and providing a theoretical reference for the sustainable development of shrimp and crab farming industries.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In a first aspect, the present invention provides a method for inducing crab hemocyte autophagy using rapamycin, comprising the following steps:

[0007] The crabs are treated with rapamycin solution; the hemolymph of the crabs is collected and prepared into a blood cell suspension; the blood cell suspension is prepared into a blood cell detection solution for the autophagy level detection.

[0008] In some other embodiments, before treating the crab with the rapamycin solution, the method further includes disinfecting the crab body, wherein the disinfection comprises wiping the base of the last pair of swimming feet of the crab body with an alcohol cotton ball.

[0009] In some other embodiments, the concentration of the rapamycin solution is 1 to 2 mg ml -1 ; Preferably, the concentration of the rapamycin solution is 1.5 mg ml -1 ;

[0010] The preparation method of the rapamycin solution is as follows: dissolving rapamycin in dimethyl sulfoxide (DMSO) to form a rapamycin stock solution, and then diluting the rapamycin stock solution with physiological saline to obtain the rapamycin solution.

[0011] The treatment conditions are: temperature of 21-26°C, treatment time of 6-48h;

[0012] Preferably, the treatment conditions are: temperature of 23±1° C. and treatment time of 24 h.

[0013] In some other embodiments, the hemolymph collection method is: wiping the base of the last pair of swimming legs of the crab body with an alcohol cotton ball; taking hemolymph from the base of the last pair of swimming legs of the crab; mixing the hemolymph with an anticoagulant, and filtering through a cell sieve to obtain crab hemolymph; wherein the volume ratio of the hemolymph to the anticoagulant is 1:(1-3);

[0014] In some other embodiments, before collecting hemolymph, the crab body is further disinfected, and the disinfection is performed by wiping the base of the last pair of swimming feet of the crab body with an alcohol cotton ball.

[0015] The composition of the anticoagulant is: 3-5 mg ml -1 , sodium citrate 5~10mg·ml -1 , Glucose 15~25mg·ml -1 ;

[0016] Preferably, the composition of the anticoagulant is: 4.2 mg ml -1 , sodium citrate 8mg·ml -1 , glucose 20.6mg·ml -1 .

[0017] In some other embodiments, the preparation method of the blood cell suspension is: centrifuging the hemolymph and removing the supernatant to obtain a cell precipitate; resuspending the cell precipitate using a PBS buffer to obtain a cell resuspension; mixing the PBS buffer with the cell resuspension; repeating the centrifugation, removing the supernatant, and resuspending steps 1 to 2 times to obtain a blood cell suspension; preferably, the density of blood cells in the blood cell suspension is ≥ 1×10 6 Pieces / ml.

[0018] In some other embodiments, the blood cell detection fluid is a crab blood cell laser confocal microscope detection fluid and a crab blood cell flow cytometer detection fluid;

[0019] The preparation method of the crab blood cell laser confocal microscope detection solution is as follows:

[0020] The crab blood cell suspension and an equal volume of a cell nucleus fluorescent probe and an equal volume of an autophagy fluorescent probe are mixed, incubated in the dark for 30 minutes, centrifuged at 4°C and 500g for 5 minutes, discarded the supernatant, resuspended in PBS, and centrifuged 1 to 2 times to obtain a crab blood cell laser confocal microscope detection solution;

[0021] The preparation method of the crab blood cell flow cytometer detection liquid is as follows: the crab blood cell suspension is mixed with equal volumes of a cell nucleus fluorescent probe, an autophagy fluorescent probe, a lysosome green fluorescent probe, and a ROS fluorescent probe, respectively, incubated in the dark for 30 minutes, centrifuged at 4°C and 500g for 5 minutes, discarded the supernatant, resuspended in PBS, and centrifuged repeatedly 1 to 2 times to obtain the crab blood cell flow cytometer detection liquid.

[0022] In some other embodiments, the cell nuclear fluorescent probe is Hoechst 33342 cell nuclear fluorescent probe;

[0023] The autophagy fluorescent probe is Cyto-ID;

[0024] The lysosomal green fluorescent probe is Lyso-Tracker Green;

[0025] The ROS fluorescent probe is H2DCFDA.

[0026] In some other embodiments, the autophagy level detection is: using a laser confocal microscope to detect the crab blood cell laser confocal microscope detection fluid, and qualitatively detecting the blood cell autophagosome structure through the cell fluorescence intensity;

[0027] The crab blood cell flow cytometer detection solution described in the flow cytometer was used for detection, the fluorescence intensity of the blood cells was detected, and the autophagosome level, lysosome activity and relative content of ROS in the blood cells were quantitatively detected.

[0028] In a second aspect, the present invention provides a crab blood cell detection solution, which is obtained by using the method of inducing crab blood cell autophagy with rapamycin described in the first aspect.

[0029] In a third aspect, the present invention provides use of the crab blood cell detection solution of the second aspect in detecting the level of autophagosomes in crab blood cells using a laser confocal microscope or a flow cytometer.

[0030] Beneficial effects of the present invention:

[0031] (1) The present invention studies and analyzes the effect of rapamycin on inducing autophagosomes in crab blood cells under normal and ammonia nitrogen stress, and confirms the effect of inducing autophagy in blood cells by qualitative and quantitative detection of autophagosome structure, combined with lysosome activity and relative content of reactive oxygen species.

[0032] (2) The fluorescence detection method based on molecular probes of the present invention has the advantages of high sensitivity, simple operation, and non-invasive detection, and the detection results of laser confocal microscopy and flow cytometry can confirm each other.

[0033] (3) By exploring the induction of crab autophagy by rapamycin, the present invention can provide a new practical basis for improving the blood cell function of crabs and provide a theoretical reference for the sustainable development of the shrimp and crab farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0035] Figure 1 The ultrastructure of autophagosomes in blood cells in the embodiment of the present invention, wherein Control is a natural seawater culture group and injected with normal saline; NH4Cl group is an ammonia nitrogen seawater culture group and injected with normal saline; NH4Cl+Rapamycin (Rap) is an ammonia nitrogen seawater culture group and injected with rapamycin; DIC is differential interference contrast; DAPI's divergent light is blue, with a maximum absorption wavelength of 358nm and a maximum emission wavelength of 461nm; FITC's divergent light is yellow-green fluorescence, with a maximum absorption wavelength of 490nm and a maximum emission wavelength of 525nm; Merge is image fusion;

[0036] Figure 2 is the ratio of autophagic hemocytes in the embodiment of the present invention;

[0037] Figure 3 The change of lysosome activity (Lysosome average fluorescence intensity ratio) of blood cells in the embodiment of the present invention;

[0038] Figure 4 It is the average fluorescence intensity of blood cells (ROS average fluorescence intensity ratio) in the embodiment of the present invention. DETAILED DESCRIPTION

[0039] Based on the fact that there is no report on the method of inducing crab blood cell autophagy by rapamycin in the prior art, the in-depth development of related research and application is limited and insufficient to meet the needs of current work. The present invention can provide a new reference for improving crab blood cell function and improving breeding results by exploring the induction of rapamycin on crab autophagy, and provide theoretical support for environmental protection and sustainable development of aquaculture.

[0040] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0041] Example 1

[0042] A method for inducing crab hemocyte autophagy with rapamycin comprises the following steps:

[0043] Step 1, preparing a rapamycin stock solution;

[0044] 91.417 mg of rapamycin was dissolved in 10 ml of dimethyl sulfoxide to prepare a 10 mM rapamycin stock solution.

[0045] Step 2, injecting rapamycin solution into the crab;

[0046] Dilute the 10 mM rapamycin stock solution to 1.5 mg ml with saline. -1 of rapamycin solution; wipe the base of the last pair of swimming legs of the crab with an alcohol cotton ball; inject the rapamycin solution into the body of the crab using a micro syringe; and place the injected crabs in normal seawater and seawater rich in ammonia nitrogen for treatment.

[0047] Wherein, the concentration of rapamycin is 1.5 mg ml -1 , the ammonia nitrogen concentration is 15 mg·L -1 .

[0048] Step 3, collecting hemolymph and preparing a suspension;

[0049] The crab hemolymph collection comprises the following steps:

[0050] Take 50 ml of anticoagulant in a centrifuge tube and centrifuge for 24 hours at 4°C for later use; wipe the base of the last pair of swimming legs of the crab with an alcohol cotton ball, and use a microsyringe to extract the hemolymph of the three-tuberculate swimming crab treated in normal and ammonia-rich seawater in step 2 respectively; mix the hemolymph and anticoagulant, and filter with a 70μm cell sieve to obtain the crab hemolymph.

[0051] The volume ratio of hemolymph to anticoagulant is 1:(1-3).

[0052] The formula of the anticoagulant: sodium chloride 4.2mg ml -1 , sodium citrate 8mg·ml -1 , glucose 20.6mg·ml -1 .

[0053] The hemolymph is made into a blood cell suspension, and the step of making the hemolymph into a blood cell suspension comprises the following steps:

[0054] The hemolymph of the crab is centrifuged at 4°C and 500g for 15 minutes, and the supernatant is discarded; the cell pellet is resuspended with an anticoagulant; different centrifuge tubes are weight-balanced and centrifuged again at 4°C and 500g for 15 minutes, and the supernatant is discarded; the cell pellet is resuspended with PBS buffer and the centrifugation operation steps are repeated 1 to 2 times; the obtained cell pellet is resuspended in PBS buffer to prepare a cell suspension to be tested.

[0055] Check the cell density of crab blood cell suspension to ensure that the final cell density is ≥1×10 6 Pieces / mL;

[0056] The method for detecting the cell density of the crab blood cell suspension comprises the following steps:

[0057] Pipette 10 μl of the blood cell suspension into a double-chamber cell counting slide, shake gently to make it evenly distributed, let it stand for 1 min, and use a whole-cell automatic counter to detect the cell density; add PBS buffer to make the final cell density ≥1×10 6 Pieces / ml.

[0058] Step 4: Prepare blood cell test solution

[0059] (1) The blood cell suspension in step 3 and an equal volume of the nuclear fluorescent probe are pipetted into a centrifuge tube, an equal volume of the autophagy fluorescent probe is added, mixed, incubated in the dark for 30 minutes, centrifuged at 4°C and 500g for 5 minutes, the supernatant is discarded, the suspension is resuspended in PBS, and the centrifugation is repeated 1 to 2 times to obtain a crab blood cell laser confocal microscopy detection solution;

[0060] (2) The blood cell suspension and equal volumes of autophagy fluorescent probe, lysosome green fluorescent probe, and ROS fluorescent probe are respectively drawn into a centrifuge tube, mixed, incubated in the dark for 30 minutes, centrifuged at 4°C and 500g for 5 minutes, the supernatant is discarded, the suspension is resuspended in PBS, and the centrifugation is repeated 1 to 2 times to obtain a crab blood cell flow cytometer detection solution;

[0061] The cell nuclear fluorescent probe is Hoechst 33342 cell nuclear fluorescent probe;

[0062] The autophagy fluorescent probe is Cyto-ID;

[0063] The lysosomal green fluorescent probe is Lyso-Tracker Green;

[0064] The ROS fluorescent probe is H2DCFDA.

[0065] Step 5, detection of crab blood cell autophagy level:

[0066] (1) using a laser confocal microscope to detect the blood cell laser confocal microscope detection fluid of the above crab, detecting the fluorescence intensity of the blood cells, and qualitatively detecting the level of blood cell autophagosomes;

[0067] Here are the steps:

[0068] The cell position is located by exciting the DAPI fluorescence channel, the cell structure integrity is determined by exciting the pseudo-bright field fluorescence channel, and the fluorescence intensity of blood cells is observed by exciting the FITC or ECD fluorescence channels to qualitatively determine the level of blood cell autophagosomes.

[0069] (2) using the above crab blood cell flow cytometer detection solution to detect the fluorescence intensity of blood cells, and quantitatively detecting the level of autophagosomes, lysosome activity and relative content of reactive oxygen species in blood cells;

[0070] Here are the steps:

[0071] The coordinate threshold was determined by forward scattered light FSC and side scattered light SSC excitation; the number of detected cells was set to 10 based on the cell aggregation results in the FSC-SSC dot plot. 5 Start the "record" mode of the flow cytometer to circle the blood cell subpopulations; excite through fluorescent channels such as FITC or ECD, and set the number of cells to be detected to 10 4 The “recording” mode of the flow cytometer was started to detect the fluorescence intensity of blood cells and the autophagosome level of blood cells was quantitatively detected.

[0072] Test results

[0073] 1. Use a 10μl pipette to draw 20μl of the Swimming Crab hemocyte autophagy laser confocal microscope detection solution onto a glass slide and cover it with a coverslip to analyze its staining.

[0074] Test results such as Figure 1 As shown in the figure, through the DAPI channel, it can be seen that in high-ammonia nitrogen seawater, the blood cells in the normal saline group emit obvious green fluorescence and have a large number of dot-like structures. Green fluorescence can be observed in the blood cells of the rapamycin injection group, but the fluorescence intensity is low and there are almost no green dot-like structures. This shows that rapamycin can reduce the accumulation of autophagosomes by activating autophagy.

[0075] 2. Use flow cytometry to detect the autophagy cytometer detection fluid of blood cells of swimming crab three tuberculosis and quantitatively detect the level of blood cell autophagosomes.

[0076] Test results such as Figure 2 As shown, under ammonia nitrogen stress, the proportion of autophagic hemocytes in the swimming crab (P. trituberculatus) injected with rapamycin was significantly lower than that in the group injected with normal saline, and this result was consistent with the rule of test result 1.

[0077] 3. Use flow cytometry to detect the lysosome flow cytometer detection fluid of the blood cells of the swimming crab three tuberculosis and quantify the lysosomal activity.

[0078] Test results such as Figure 3 As shown, under ammonia nitrogen stress, the lysosomal activity of hemocytes in the group injected with rapamycin was significantly higher than that in the group injected with normal saline.

[0079] 4. Use flow cytometry to detect ROS flow cytometer detection fluid of blood cells of swimming crab three tuberculosis and quantify ROS level.

[0080] Test results such as Figure 4 As shown, under ammonia nitrogen stress, the ROS level in the blood cells of the swimming crab (P. trituberculatus) injected with rapamycin was significantly lower than that in the group injected with normal saline.

[0081] Based on the above analysis, the present invention can improve the function of crab blood cells by exploring the induction of crab autophagy by rapamycin, and provide a theoretical reference for improving the effectiveness of shrimp and crab farming and promoting the sustainable development of the industry. In addition, the fluorescence detection method based on molecular probes of the present invention has the advantages of high sensitivity, simple operation, and non-invasive detection, and the detection results of laser confocal microscopy and flow cytometry can confirm each other.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for inducing crab blood cell autophagy with rapamycin, characterized in that: The following steps are involved: The crabs are treated with rapamycin solution; the hemolymph of the crabs is collected and prepared into a blood cell suspension; the blood cell suspension is prepared into a blood cell detection solution for the autophagy level detection.

2. The method for inducing crab blood cell autophagy with rapamycin according to claim 1, characterized in that: The concentration of the rapamycin solution is 1-2 mg ml -1 ; Preferably, the concentration of the rapamycin solution is 1.5 mg ml -1 ; Alternatively, the preparation method of the rapamycin solution is: dissolving rapamycin in dimethyl sulfoxide to form a rapamycin stock solution, and then diluting the rapamycin stock solution with physiological saline to obtain the rapamycin solution.

3. The method for inducing crab blood cell autophagy with rapamycin according to claim 1, characterized in that: The treatment conditions are: temperature of 21-26°C, treatment time of 6-48h; Preferably, the treatment conditions are: temperature of 23±1° C. and treatment time of 24 h.

4. The method for inducing crab hemocyte autophagy with rapamycin according to claim 1, characterized in that: The hemolymph collection method is as follows: hemolymph is collected from the base of the last pair of swimming legs of the crab; the hemolymph is mixed with an anticoagulant, and the crab hemolymph is obtained after filtering through a cell sieve; wherein the volume ratio of the hemolymph to the anticoagulant is 1:(1-3); The composition of the anticoagulant is: 3-5 mg ml -1 , sodium citrate 5~10mg·ml -1 , Glucose 15~25mg·ml -1 ; Preferably, the composition of the anticoagulant is: 4.2 mg ml -1 , sodium citrate 8mg·ml -1 , glucose 20.6mg·ml -1 .

5. The method of inducing crab hemocyte autophagy with rapamycin according to claim 1, characterized in that: The preparation method of the blood cell suspension comprises: centrifuging the hemolymph and removing the supernatant to obtain a cell precipitate; resuspending the cell precipitate with a PBS buffer to obtain a cell resuspension; mixing the PBS buffer with the cell resuspension; repeating the centrifugation, removing the supernatant, and resuspending steps 1 to 2 times to obtain a blood cell suspension; preferably, the density of blood cells in the blood cell suspension is ≥ 1×10 6 Pieces / mL.

6. The method for inducing crab hemocyte autophagy with rapamycin according to claim 1, characterized in that: The blood cell detection solution is: crab blood cell laser confocal microscope detection solution and crab blood cell flow cytometer detection solution; The preparation method of the crab blood cell laser confocal microscope detection solution is as follows: The crab blood cell suspension and an equal volume of a cell nucleus fluorescent probe and an equal volume of an autophagy fluorescent probe are mixed, incubated in the dark for 30 minutes, centrifuged at 4°C and 500g for 5 minutes, discarded the supernatant, resuspended in PBS, and centrifuged 1 to 2 times to obtain a crab blood cell laser confocal microscope detection solution; The preparation method of the crab blood cell flow cytometer detection liquid is as follows: the crab blood cell suspension is mixed with equal volumes of a cell nucleus fluorescent probe, an autophagy fluorescent probe, a lysosome green fluorescent probe, and a ROS fluorescent probe, respectively, incubated in the dark for 30 minutes, centrifuged at 4°C and 500g for 5 minutes, discarded the supernatant, resuspended in PBS, and centrifuged repeatedly 1 to 2 times to obtain the crab blood cell flow cytometer detection liquid.

7. The method for inducing crab blood cell autophagy with rapamycin according to claim 6, characterized in that: The cell nuclear fluorescent probe is Hoechst 33342 cell nuclear fluorescent probe; The autophagy fluorescent probe is Cyto-ID; The lysosomal green fluorescent probe is Lyso-Tracker Green; The ROS fluorescent probe is H2DCFDA.

8. The method for inducing crab blood cell autophagy with rapamycin according to claim 1, characterized in that: The autophagy level detection is: using a laser confocal microscope to detect the crab blood cell laser confocal microscope detection fluid, and qualitatively detecting the blood cell autophagosome structure through the cell fluorescence intensity; The crab blood cell flow cytometer detection solution described in the flow cytometer was used for detection, the fluorescence intensity of the blood cells was detected, and the autophagosome level, lysosome activity and relative content of ROS in the blood cells were quantitatively detected.

9. A crab blood cell detection solution, characterized in that: The method for inducing crab hemocyte autophagy with rapamycin according to any one of claims 1 to 8 is used to obtain the product.

10. Use of the crab blood cell detection solution according to claim 9 in detecting the level of autophagosomes in crab blood cells using a laser confocal microscope or a flow cytometer.

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