Method for extracting, purifying and identifying high-purity fish liver lipid droplet mitochondria

Abstract

The invention relates to a method for extracting, purifying and identifying high-purity fish liver surrounding lipid droplet mitochondria, and belongs to the technical field of animal biology.The method comprises the following steps that S1, fish gill cutting, bloodletting, liver taking, cleaning and impurity removal are conducted, the washed liver is cut into pieces, grinding is conducted, and then unbroken cells and tissue blocks are removed through centrifugation; and S2, performing low-speed centrifugation to separate the fat layer, and then performing high-speed centrifugation to separate mitochondria from the fat layer. According to the method for extracting, purifying and identifying the high-purity fish liver lipid droplet mitochondria, the purification and identification method is used for extracting the fish liver lipid droplet mitochondria, the operation is simple and easy to implement, the extraction efficiency is high, and PDM impurities extracted by utilizing tissues are few; according to the present invention, the cell debris problem existing in the existing method is completely removed by performing multiple differential centrifugation, the cytoplasm pollution is reduced by using multiple resuspension steps in the differential centrifugation, and the contained impurities are diluted by continuously expanding the resuspension ratio so as to improve the purity.

Description

Technical Field

[0001] The invention relates to the field of animal biotechnology, in particular to a method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria. Background Art

[0002] The main characteristics of the current aquaculture model are high fat content in feed, high stocking density, and unbalanced feeding, which can easily cause excessive deposition of fish fat, especially in the liver, and may even cause fatty liver, which seriously affects the growth rate and health of fish; at the same time, it also affects the quality of fish muscle, resulting in poor taste for consumers. Therefore, exploring the formation mechanism of fatty liver has also become a research hotspot in the current aquaculture industry, but to date there has been no breakthrough in the formation mechanism of fish fatty liver. The reason is that fish liver fat deposition is based on a network structure of multiple molecules and proteins, which is characterized by a complex network with multiple levels and multiple tissue forms, making it difficult to find key targets for regulating fatty liver.

[0003] Research on mammalian organelles has shown that the organelle level may be a breakthrough in revealing the formation mechanism of fatty liver. Lipid droplets are the main storage site for intracellular lipids; and mitochondria are the main site for fat decomposition. Studies have pointed out that there is mutual contact and interaction between lipid droplets and mitochondria, which are called perilipid droplet mitochondria, and PDM is crucial to the formation process of fat deposition, which is a key target for clarifying the formation mechanism of fatty liver.

[0004] However, the successful separation and extraction of PDM is the basis for the above research. Unfortunately, there is no mature method for the extraction, purification and identification of fish PDM. Therefore, we used yellow catfish as a model and proposed a method for the extraction, purification and identification of fish liver PDM based on differential centrifugation for the first time. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria. The extracted PDM has a complete structure, has been verified to have high purity, and is suitable for frozen sample extraction. Pre-cooling equipment and solutions are used throughout the extraction process.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria, comprising the following steps:

[0007] S1, take the fish, cut the gills, bleed, take the liver, clean and remove impurities, cut the washed liver into pieces, grind it and centrifuge it to remove unbroken cells and tissue blocks;

[0008] S2. Centrifuge at low speed to separate the fat layer and then centrifuge at high speed to separate the mitochondria from the fat layer.

[0009] Furthermore, the specific method in S1 is:

[0010] S1-1. At room temperature, select 200-500g yellow catfish, wipe the surface of the fish with 75% alcohol, and dissect it in a sterile environment.

[0011] S1-2, take the liver and soak it in 75% alcohol for 30 seconds;

[0012] S1-3. Wash the liver three times with ice-cold PBS to remove connective tissue and blood as much as possible.

[0013] Furthermore, the specific method for obtaining the liver tissue block in S1 is to add 1 ml of mitochondrial isolation buffer MIB per 100 mg of tissue to the washed liver tissue and cut it into 1 mm 3 ;

[0014] Furthermore, the specific method of grinding the liver tissue blocks in S1 is to transfer the chopped liver tissue and MIB into a grinding tube, slowly and gently homogenize to avoid damaging mitochondria, until there are no tissue lumps, and then centrifuge at 500g and 4°C for 5min to remove crude impurities. The whole process is performed on ice.

[0015] Furthermore, the mitochondrial isolation buffer MIB is prepared by mixing 250mmol / L sucrose, 50mmol / L HEPES and 2mmol / L EGTA. After mixing, the pH is adjusted to 7.4 and stored at 4°C.

[0016] Furthermore, the specific process in S2 is:

[0017] S2-1, after centrifugation to remove tissue blocks, the homogenate is subjected to ultrasonic disruption at a frequency of 20-25% and a disruption time of 3-5 min;

[0018] S2-2, after crushing, centrifuge the homogenate at 900g at 4°C for 10 min to remove the precipitate, carefully separate the layers, and aspirate the upper fat layer without disturbing the lower layer;

[0019] S2-3. After the fat layer is removed by centrifugation, the homogenate is subjected to ultrasonic disruption at a frequency of 20-25% and a disruption time of 3-5 min. The homogenate is centrifuged 2-3 times to obtain a high-purity fat layer.

[0020] Further, the specific operation after low-speed centrifugation in S2 is:

[0021] A1. Collect the fat layer, resuspend it with MIB+BSA, centrifuge at 9000g, 4℃ for 10min, and resuspend the precipitate with MRB according to the tissue content, with a ratio of 1:6;

[0022] A2. The resuspension was subjected to differential centrifugation. First, centrifuge at 900g and 4°C for 10 min, collect the supernatant, centrifuge at 9000g and 4°C for 20 min, remove the supernatant, and resuspend the precipitate in MRB according to the tissue content, at a ratio of 1:5;

[0023] A3, centrifuge at 900g, 4℃ for 10min, collect the supernatant, centrifuge at 9000g, 4℃ for 20min, discard the supernatant, and resuspend the precipitate in MRB according to the tissue content, at a ratio of 1:4;

[0024] A4. Finally, centrifuge at 9000g and 4℃ for 20min, remove the supernatant, and the precipitate is the perilipid droplet mitochondria.

[0025] Furthermore, the MIB+BSA in A1 is prepared by mixing 250mmol / L sucrose, 50mmol / L HEPES, 2mmol / L EGTA and 2% BSA. After mixing, the pH is adjusted to 7.4 and stored at 4°C; the MRB in A1 is prepared by mixing 250mmol / L mannitol, 5mmol / L HEPES and 0.5mmol / L EDTA. After mixing, the pH is adjusted to 7.4 and stored at 4°C.

[0026] Compared with the prior art, the present invention provides a method for extracting, purifying and identifying mitochondria from fish liver perilipid droplets with high purity, which has the following beneficial effects:

[0027] 1. The method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria is simple and easy to operate, with high extraction efficiency and less PDM impurities in tissue extraction by adopting the above purification and identification method to extract fish liver perilipid droplet mitochondria.

[0028] 2. The method for extracting, purifying and identifying mitochondria from perilipid droplets of fish liver with high purity performs multiple differential centrifugation to fully remove the cell debris problem existing in the existing method, reduces cytoplasmic contamination by utilizing multiple resuspension steps in differential centrifugation, and continuously increases the resuspension ratio to dilute the impurities contained, thereby improving purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The main process steps of the extraction process of perilipid droplets mitochondria in the liver of yellow catfish in the method for extracting, purifying and identifying high-purity perilipid droplets mitochondria in the liver of fish of the present invention;

[0030] Figure 2 The invention discloses a method for extracting, purifying and identifying mitochondria of perilipid droplets in fish livers with high purity, and extracting perilipid droplets of yellow catfish livers by transmission electron microscope detection;

[0031] Figure 3Fluorescence confocal verification of the purification effect of the extracted perilipid droplet mitochondria of a high-purity fish liver perilipid droplet mitochondria of the present invention;

[0032] Figure 4 The invention discloses a method for extracting, purifying and identifying mitochondria of perilipid droplets of fish liver with high purity, and uses an immunoblotting method to verify the purification effect of the extracted perilipid droplet mitochondria. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention and the accompanying drawings 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.

[0034] See also Figures 1 to 4 In this embodiment, a method for extracting, purifying and identifying high-purity fish liver perilipid mitochondria comprises the following steps:

[0035] (1) Select a yellow catfish of appropriate size, cut its gills, bleed it, and remove its liver. The liver is then washed 2-3 times in PBS.

[0036] (2) Add the washed liver to pre-cooled MIB, wash 3-4 times to remove blood, chop and remove.

[0037] (3) Add 1 ml of MIB for every 100 mg of tissue and homogenize slowly and gently to avoid damaging mitochondria until there are no tissue lumps. After grinding, centrifuge at 500 g for 5 min to remove crude impurities.

[0038] (4) The homogenate after centrifugation is subjected to ultrasonic disruption at a frequency of 20-25% and a disruption time of 3-5 min.

[0039] (5) After disruption, the homogenate is centrifuged at 900 g for 10 min to remove the precipitate, and the layers are carefully separated. The upper fat layer is aspirated without disturbing the lower layer. The fat layer is centrifuged 2-3 times under the above conditions to obtain a high-purity fat layer.

[0040] (6) Collect the fat layer, resuspend it with MIB+BSA, centrifuge it at 9000g for 10 min, and resuspend the precipitate with MRB according to the tissue content at a ratio of 1:6; use differential centrifugation for the resuspension, first centrifuge it at 900g for 10 min, collect the supernatant, centrifuge it at 9000g for 20 min, remove the supernatant, and resuspend the precipitate with MRB according to the tissue content at a ratio of 1:5; centrifuge it at 900g for 10 min, collect the supernatant, centrifuge it at 9000g for 20 min, remove the supernatant, and resuspend the precipitate with MRB according to the tissue content at a ratio of 1:4; finally centrifuge it at 9000g for 20 min, remove the supernatant, and the precipitate is the perilipid droplet mitochondria.

[0041] Specifically, the liver tissue blocks in S1 were obtained by adding 1 ml of mitochondrial isolation buffer (MIB) per 100 mg of the washed liver tissue and cutting it into 1 mm pieces. 3 .

[0042] Specifically, the specific method of grinding the liver tissue blocks in S1 is to transfer the chopped liver tissue and MIB into a grinding tube, slowly and gently homogenize to avoid damaging mitochondria, until there are no tissue lumps, and then centrifuge at 500g and 4°C for 5min to remove crude impurities. The whole process is performed on ice.

[0043] Specifically, MIB contains sucrose 250 mmol / L, HEPES 50 mmol / L, and EGTA 2 mmol / L.

[0044] Specifically, MIB+BSA contains sucrose 250 mmol / L, HEPES 50 mmol / L, EGTA 2 mmol / L, and BSA 2%.

[0045] Specifically, MRB contains mannitol 250 mmol / L, HEPES 5 mmol / L, EDTA 0.5 mmol / L. All reagents are adjusted to pH 7.4 and stored at 4°C.

[0046] The purity of PDM extracted in Example 1 was detected by electron microscopy, immunofluorescence and Western blot.

[0047] The results are as follows Figures 2 to 4 As shown, Figure 2 Electron microscopic photos show that most of the crude mitochondria extracted using this method have clear mitochondrial cristae structure; pure mitochondria have complete cristae structure and few impurities; PDM is surrounded by lipid droplets, without contamination by other organelles, and there are clusters, indicating that the peri-lipid droplet mitochondria extracted by the present invention are characterized by high purity and large quantity. Figure 3The immunofluorescence photos of Zhongli showed that the mitochondria in the extract were tightly wrapped around the surface of lipid droplets and were clustered, indicating that the mitochondria surrounding lipid droplets in the extract were of high purity and large number. Figure 4 The expression of different organelle marker proteins in different organelle extracts was detected by immunoblotting. The PDM extracted by the present invention had basically no PLIN2, TGN46, and GAPDH expression, indicating that the extracted product had no cytoplasm, lipid droplet and Golgi apparatus contamination, and the expression levels of the remaining marker proteins were consistent with the protein localization and were consistent with the description in the literature, proving that the mitochondria of the perilipid droplets of the liver of the yellow catfish extracted by this method were basically free of impurity contamination and had the characteristics of high purity.

[0048] It should be noted that Figure 2 PDM in the figure stands for perimitochondrial mitochondria, MT stands for mitochondria, and LD stands for lipid droplets.

[0049] It should be noted that Figure 3 The green color represents lipid droplets, the red color represents mitochondria, and the yellow color represents perilipid droplet mitochondria.

[0050] It should be noted that Figure 4 PDM in the figure is the perilipid droplet mitochondria, CM cytoplasm is the mitochondria, and Cyto is the cytoplasm. TGN46 is located in the Golgi apparatus and is a Golgi apparatus marker protein; PLIN2 is related to lipid droplet synthesis in lipid droplets and is located on lipid droplets; GAPDH is a cytoplasmic marker protein and is expressed in large quantities in the cytoplasm; TOM20 is a mitochondrial marker protein and is located in the inner membrane of mitochondria.

[0051] The beneficial effects of the present invention are:

[0052] The method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria uses the purification and identification method to extract fish liver perilipid droplet mitochondria, which is simple and easy to operate, has high extraction efficiency, and uses less PDM impurities extracted from tissues.

[0053] The method for extracting, purifying and identifying mitochondria from fish liver perilipid droplets with high purity performs multiple differential centrifugation to fully remove the cell debris problem existing in the existing method, reduces cytoplasmic contamination by utilizing multiple resuspension steps in differential centrifugation, and continuously increases the resuspension ratio to dilute the impurities contained, thereby improving purity.

[0054] The specific embodiments provided in the present invention are for clearer explanation. The relevant technical personnel may make reasonable modifications to the corresponding steps in the specific operation process according to the experimental conditions. The materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting, purifying and identifying high-purity fish liver perilipid mitochondria, characterized in that: The following steps are involved: S1, take the fish, cut the gills, bleed, take the liver, clean and remove impurities, cut the washed liver into pieces, grind it and centrifuge it to remove unbroken cells and tissue blocks; S2. Centrifuge at low speed to separate the fat layer and then centrifuge at high speed to separate the mitochondria from the fat layer.

2. The method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria according to claim 1, characterized in that: The specific method in S1 is: S1-1. At room temperature, select 200-500g yellow catfish, wipe the surface of the fish with 75% alcohol, and dissect it in a sterile environment. S1-2, take the liver and soak it in 75% alcohol for 30 seconds; S1-3. Wash the liver three times with ice-cold PBS to remove connective tissue and blood as much as possible.

3. The method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria according to claim 1, characterized in that: The specific method for obtaining liver tissue blocks in S1 is to add 1 ml of mitochondrial isolation buffer MIB per 100 mg of tissue to the washed liver tissue and cut it into 1 mm 3 .

4. The method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria according to claim 1, characterized in that: The specific method of grinding liver tissue blocks in S1 is to transfer the chopped liver tissue and MIB into a grinding tube, slowly and gently homogenize to avoid damaging mitochondria, until there are no tissue lumps, and then centrifuge at 500g and 4℃ for 5min to remove crude impurities. The whole process is performed on ice.

5. The method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria according to claim 3, characterized in that: The mitochondrial isolation buffer MIB is prepared by mixing 250 mmol / L sucrose, 50 mmol / L HEPES and 2 mmol / L EGTA. After mixing, the pH is adjusted to 7.4 and stored at 4°C.

6. The method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria according to claim 1, characterized in that: The specific process in S2 is: S2-1, after centrifugation to remove tissue blocks, the homogenate is subjected to ultrasonic disruption at a frequency of 20-25% and a disruption time of 3-5 min; S2-2, after crushing, centrifuge the homogenate at 900g at 4°C for 10 min to remove the precipitate, carefully separate the layers, and aspirate the upper fat layer without disturbing the lower layer; S2-3. After the fat layer is removed by centrifugation, the homogenate is subjected to ultrasonic disruption at a frequency of 20-25% and a disruption time of 3-5 min. The homogenate is centrifuged 2-3 times to obtain a high-purity fat layer.

7. The method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria according to claim 1, characterized in that: The specific operations after S2 low-speed centrifugation are: A1. Collect the fat layer, resuspend it with MIB+BSA, centrifuge at 9000g, 4℃ for 10min, and resuspend the precipitate with MRB according to the tissue content, with a ratio of 1:6; A2. The resuspension was subjected to differential centrifugation. First, centrifuge at 900g and 4°C for 10 min, collect the supernatant, centrifuge at 9000g and 4°C for 20 min, remove the supernatant, and resuspend the precipitate in MRB according to the tissue content, at a ratio of 1:5; A3, centrifuge at 900g, 4℃ for 10min, collect the supernatant, centrifuge at 9000g, 4℃ for 20min, discard the supernatant, and resuspend the precipitate in MRB according to the tissue content, with a ratio of 1:4; A4. Finally, centrifuge at 9000g and 4℃ for 20min, remove the supernatant, and the precipitate is the perilipid droplet mitochondria.

8. The method for extracting, purifying and identifying high-purity fish liver perilipid droplet mitochondria according to claim 7, characterized in that: The MIB+BSA in A1 is prepared by mixing 250mmol / L sucrose, 50mmol / L HEPES, 2mmol / L EGTA and 2% BSA. After mixing, the pH is adjusted to 7.4 and the mixture is stored at 4°C. The MRB in A1 is prepared by mixing 250mmol / L mannitol, 5mmol / L HEPES and 0.5mmol / L EDTA. After mixing, the pH is adjusted to 7.4 and the mixture is stored at 4°C.

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