Isolated culture and induced differentiation method of grass carp precursor fat cells

Abstract

The invention relates to an isolated culture and induced differentiation method of grass carp precursor adipocytes, and belongs to the technical field of adipocyte isolated culture, and the method comprises the following steps: S1, taking grass carp adipose tissues under an aseptic condition, and digesting the tissues with I-type collagenase digestive juice for 30-60 min to obtain grass carp precursor adipocytes; s2, inoculating the digested grass carp precursor fat cells into a 24-hole cell plate, putting the 24-hole cell plate into a 28 DEG C cell culture box for culture, and utilizing a grass carp precursor fat cell culture medium for culture until the cells are adhered to the wall. According to the isolated culture and induced differentiation method of the grass carp precursor fat cells, an enzymolysis method for simplifying primary culture of fish precursor tissue cells is combined with a tissue crushing method and a collagen enzymolysis method, and the precursor fat cells are purified by differential centrifugation; by adjusting the formula of the culture medium, the pH stability of the culture medium can be better maintained, and by adjusting the cell osmotic pressure, the phenomenon that a large number of cells die due to internal and external osmotic pressure difference during digestion can be avoided.

Description

Technical Field

[0001] The invention relates to the technical field of separation and culture of fat cells, and in particular to a method for separation and culture of grass carp precursor fat cells and induction differentiation thereof. Background Art

[0002] Currently, in the aquaculture industry, the problem of "big belly" of fish occurs frequently, and its essence is the excessive deposition of fish fat. Excessive deposition of fat can easily reduce the disease resistance of fish, induce large-scale infectious fish diseases, and even lead to mass deaths; at the same time, it also reduces the muscle quality of fish, affects the taste of consumers, and seriously affects the benefits of aquaculture. The fundamental reason for fat accumulation is the excessive proliferation, differentiation and maturation of preadipocytes. Therefore, if we want to reveal the mechanism of excessive fat deposition in fish, we must clarify the proliferation, differentiation and maturation process of fish preadipocytes, and the successful isolation and cultivation of fish preadipocytes and the establishment of mature induced differentiation methods are the cornerstones of the above research.

[0003] However, the current method of fish preadipocytes and induced differentiation is not mature, and only a patent for the separation and culture of yellow croaker preadipocytes and its induction method has been published (CN109182256B). However, yellow croaker is a marine fish, and its living environment is very different from that of freshwater fish, so this method cannot be directly applied to the preadipocytes and induced differentiation process of freshwater fish.

[0004] Grass carp is one of the four major freshwater fishes in China, accounting for 16.83% of the total freshwater fish production. However, the problem of excessive fat also plagues the healthy farming of grass carp. Therefore, in order to solve the above industrial problems, it is necessary to use grass carp as a research object and develop a method for isolating and culturing preadipocytes suitable for freshwater fish and inducing differentiation. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for isolating and culturing grass carp precursor adipocytes and inducing their differentiation, which has the advantages of simplicity, good repeatability, low cost and rapid cell growth, and can provide an effective in vitro cell model for studying the mechanism of excessive fat accumulation in grass carp.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for isolating and culturing grass carp preadipocytes, comprising the following steps:

[0007] S1. Under sterile conditions, grass carp adipose tissue was obtained and digested with type I collagenase digestion solution for 30-60 min to obtain grass carp preadipocytes;

[0008] S2. The digested grass carp preadipocytes were inoculated into a 24-well cell plate, placed in a cell culture incubator at 28° C., and cultured using grass carp preadipocyte culture medium until the cells adhered to the wall.

[0009] Further, the specific method of obtaining grass carp adipose tissue in S1 is:

[0010] A1. At room temperature, select a grass carp of 1-1.5 kg in size, wipe the surface of the fish with 75% alcohol, and dissect it in a sterile environment.

[0011] A2. Take mesenteric fat tissue and soak it in 75% alcohol for 30 seconds;

[0012] A3. Wash the fat three times with ice-cold PBS (pH 7.4) in a clean bench to remove connective tissue and blood as much as possible;

[0013] A4. Wash the grass carp fat tissue blocks with DMEM / F12 culture medium.

[0014] Furthermore, the specific digestion method of grass carp adipose tissue in S1 is:

[0015] B1. Place the washed adipose tissue in a culture dish containing 2-3 times the volume of type I collagenase digestion solution and cut it into pieces of about 1 mm3;

[0016] B2. Transfer the chopped adipose tissue and collagenase digestion solution into a 50 ml centrifuge tube and place it on a shaker at 28°C for 30-60 minutes;

[0017] B3. When there are no obvious tissue blocks in the centrifuge tube, terminate the digestion with a culture medium containing 10% fetal bovine serum;

[0018] Furthermore, the preparation method of the type I collagenase digestion solution in S1 is to add type I collagenase, bovine serum albumin, penicillin and streptomycin to DMEM / F12 culture medium containing calcium and magnesium ions; wherein the concentration of type I collagenase is 1 mg / ml, 2% bovine serum albumin, the concentration of penicillin is 100 IU / mL, and the concentration of streptomycin is 100 μg / mL.

[0019] Further, the specific steps in S1 are:

[0020] S1-1. After the digestion of the culture medium is terminated, centrifuge at 2000 rpm for 10 min, discard the upper mature adipocytes and supernatant, add 2-3 times the volume of red blood cell lysis buffer, and lyse for 2 min;

[0021] S1-2, after lysis, neutralize with DMEM / F12 medium containing 10% fetal bovine serum. After neutralization, filter twice with a 200-mesh cell sieve, and centrifuge the obtained cell suspension at 3000 rpm for 10 min.

[0022] S1-3. After centrifugation, discard the supernatant and the lower layer of red blood cells, wash twice with fresh DMEM / F12 medium, resuspend the cells with growth medium DMEM / F12, 10% FBS, 1% double antibody, and culture in a 24-well cell culture plate;

[0023] S1-4, after 24 hours of culture, the medium was changed and the cells were washed with PBS to remove non-adherent red blood cells and tissue blocks;

[0024] S1-5. After two days, change the medium. When the cells proliferate to 80-90%, they can be digested and passaged.

[0025] Furthermore, the culture medium for preadipocytes in S2 was DMEM / F12 medium containing 10 vol% fetal bovine serum, 3.57 mg / ml HEPES, 100 IU / mL penicillin and 100 μg / mL streptomycin.

[0026] Another technical problem to be solved by the present invention is to provide a method for inducing differentiation of grass carp preadipocytes, comprising the following steps:

[0027] C1. Cell preparation: Grass carp preadipocytes are separated by the enzymatic hydrolysis method in S1, and the grass carp preadipocytes are cultured in serum-containing medium to ensure their normal growth and proliferation;

[0028] C2. Cell induction differentiation: When the grass carp preadipocytes grow to 80%-90% confluence in the culture dish, the induction differentiation is started; on the basis of the serum culture medium, an inducer is added to prepare an induction culture medium; the cell culture medium is replaced with the induction culture medium, and the induction differentiation process is started;

[0029] C3. Differentiation process management: During the induction differentiation period, according to the cell growth and the color change of the culture medium, replace the fresh induction medium in time to ensure that the cells are in the best growth and differentiation environment; regularly observe the morphological changes of the cells and record the key time points in the differentiation process, such as the beginning of cell differentiation and the formation of fat droplets;

[0030] C4. Completion of cell differentiation: After a period of induced differentiation, the degree of cell differentiation is evaluated by staining and microscopic observation; further experimental analysis is performed on the differentiated adipocytes according to the experimental purpose, such as detecting the expression levels of lipid metabolism-related genes and proteins, and studying the biological functions of adipocytes.

[0031] Furthermore, the serum-containing medium in C1 is prepared by adding 90 mL of DMEM / F12 medium, 10 mL of serum, and 0.25 g of NaHCO to every 100 mL of culture solution. 3 and 0.1 mL of antibiotics, also filtered through a 0.22 μm filter and stored at 4°C.

[0032] Furthermore, the inducers in C2 include insulin, dexamethasone, IBMX and a lipid mixture, and their concentrations are 20 μg / mL, 1 μmol / L, 10 nmol / L and 100 μmol / L, respectively.

[0033] Compared with the prior art, the present invention provides a method for isolating and culturing grass carp preadipocytes and inducing differentiation thereof, which has the following beneficial effects:

[0034] 1. The method for isolating and culturing grass carp precursor adipocytes and inducing their differentiation simplifies the enzymatic hydrolysis method for primary culture of fish precursor tissue cells, combines tissue disruption with collagen enzymatic hydrolysis, and uses differential centrifugation to purify precursor adipocytes; by adjusting the formula of the culture medium, the pH stability of the culture medium can be better maintained, and by adjusting the cell osmotic pressure, a large number of cell deaths due to the difference in internal and external osmotic pressures during digestion can be avoided.

[0035] 2. The method for isolating and culturing grass carp preadipocytes and inducing their differentiation can adjust the pH value of the reaction system by adding HEPES to the preadipocyte culture medium, and can control a constant pH range for a long time, which can effectively maintain the stability of cells and proteins, which is important for maintaining the stability of the internal and external environment of grass carp preadipocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The present invention is a schematic diagram of the separation of grass carp preadipocytes according to a method for the separation, culture and differentiation induction of grass carp preadipocytes.

[0037] Figure 2 This is a light microscopic image of the first generation of grass carp preadipocytes cultured in a method for isolating and culturing grass carp preadipocytes and inducing differentiation thereof according to the present invention;

[0038] Figure 3 This is a light microscopic image of the second generation of grass carp preadipocytes cultured in a method for isolating and culturing grass carp preadipocytes and inducing differentiation thereof according to the present invention;

[0039] Figure 4 This is a light microscopic image of the third generation of grass carp preadipocytes cultured in a method for isolating and culturing grass carp preadipocytes and inducing differentiation thereof according to the present invention;

[0040] Figure 5This is a light microscopic image of the fifth generation of grass carp preadipocytes cultured in a method for isolating and culturing grass carp preadipocytes and inducing differentiation thereof according to the present invention;

[0041] Figure 6 The oil red staining result of cells on the third day of isolation and culture of a method for isolating and culturing grass carp preadipocytes and inducing differentiation thereof according to the present invention;

[0042] Figure 7 The oil red staining result of the second generation of cells isolated and cultured in the method for isolating and culturing grass carp precursor adipocytes and inducing differentiation thereof of the present invention;

[0043] Figure 8 The present invention is a light microscope picture of grass carp preadipocytes induced by an adipogenic inducing agent in a method for isolating, culturing and inducing differentiation of grass carp preadipocytes. DETAILED DESCRIPTION

[0044] 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.

[0045] Overall embodiment:

[0046] See also Figures 1 to 8 A method for isolating and culturing grass carp preadipocytes in this embodiment comprises the following steps:

[0047] S1. Under sterile conditions, grass carp adipose tissue was obtained and digested with type I collagenase digestion solution for 30-60 min to obtain grass carp preadipocytes;

[0048] S2. The digested grass carp preadipocytes were inoculated into a 24-well cell plate, placed in a cell culture incubator at 28° C., and cultured using grass carp preadipocyte culture medium until the cells adhered to the wall.

[0049] Specifically, the specific method for obtaining grass carp adipose tissue in S1 is:

[0050] A1. At room temperature, select a grass carp of 1-1.5 kg in size, wipe the surface of the fish with 75% alcohol, and dissect it in a sterile environment.

[0051] A2. Take mesenteric fat tissue and soak it in 75% alcohol for 30 seconds;

[0052] A3. Wash the fat three times with ice-cold PBS (pH 7.4) in a clean bench to remove connective tissue and blood as much as possible;

[0053] A4. Wash the grass carp fat tissue blocks with DMEM / F12 culture medium.

[0054] Specifically, the specific way of digestion of grass carp adipose tissue in S1 is:

[0055] B1. Place the washed adipose tissue in a culture dish containing 2-3 times the volume of type I collagenase digestion solution and cut it into pieces of about 1 mm3;

[0056] B2. Transfer the chopped adipose tissue and collagenase digestion solution into a 50 ml centrifuge tube and place it on a shaker at 28°C for 30-60 minutes;

[0057] B3. When there are no obvious tissue blocks in the centrifuge tube, terminate the digestion with culture medium containing 10% fetal bovine serum.

[0058] Specifically, the preparation method of the type I collagenase digestion solution in S1 is to add type I collagenase, bovine serum albumin, penicillin and streptomycin to DMEM / F12 culture medium containing calcium and magnesium ions; wherein the concentration of type I collagenase is 1 mg / ml, 2% bovine serum albumin, the concentration of penicillin is 100 IU / mL, and the concentration of streptomycin is 100 μg / mL.

[0059] Specifically, the specific steps in S1 are:

[0060] S1-1. After the digestion of the culture medium is terminated, centrifuge at 2000 rpm for 10 min, discard the upper mature adipocytes and supernatant, add 2-3 times the volume of red blood cell lysis buffer, and lyse for 2 min;

[0061] S1-2, after lysis, neutralize with DMEM / F12 medium containing 10% fetal bovine serum. After neutralization, filter twice with a 200-mesh cell sieve, and centrifuge the obtained cell suspension at 3000 rpm for 10 min.

[0062] S1-3. After centrifugation, discard the supernatant and the lower layer of red blood cells, wash twice with fresh DMEM / F12 medium, resuspend the cells with growth medium DMEM / F12, 10% FBS, 1% double antibody, and culture in a 24-well cell culture plate;

[0063] S1-4, after 24 hours of culture, the medium was changed and the cells were washed with PBS to remove non-adherent red blood cells and tissue blocks;

[0064] S1-5. After two days, change the medium. When the cells proliferate to 80-90%, they can be digested and passaged.

[0065] Specifically, the culture medium for preadipocytes in S2 is DMEM / F12 medium containing 10 vol% fetal bovine serum, 3.57 mg / ml HEPES, 100 IU / mL penicillin and 100 μg / mL streptomycin.

[0066] It should be noted that the red blood cell lysis solution was purchased from Beijing Solebow Technology Co., Ltd.

[0067] Another technical problem to be solved by the present invention is to provide a method for inducing differentiation of grass carp preadipocytes, comprising the following steps:

[0068] C1. Cell preparation: Grass carp preadipocytes are separated by the enzymatic hydrolysis method in S1, and the grass carp preadipocytes are cultured in serum-containing medium to ensure their normal growth and proliferation;

[0069] C2. Cell induction differentiation: When the grass carp preadipocytes grow to 80%-90% confluence in the culture dish, the induction differentiation is started; on the basis of the serum culture medium, an inducer is added to prepare an induction culture medium; the cell culture medium is replaced with the induction culture medium, and the induction differentiation process is started;

[0070] C3. Differentiation process management: During the induction differentiation period, according to the cell growth and the color change of the culture medium, replace the fresh induction medium in time to ensure that the cells are in the best growth and differentiation environment; regularly observe the morphological changes of the cells and record the key time points in the differentiation process, such as the beginning of cell differentiation and the formation of fat droplets;

[0071] C4. Completion of cell differentiation: After a period of induced differentiation, the degree of cell differentiation is evaluated by staining and microscopic observation; further experimental analysis is performed on the differentiated adipocytes according to the experimental purpose, such as detecting the expression levels of lipid metabolism-related genes and proteins, and studying the biological functions of adipocytes.

[0072] Specifically, the serum-containing culture medium in C1 is prepared by adding 90 mL of DMEM / F12 culture medium, 10 mL of serum, 0.25 g of NaHCO 3 and 0.1 mL of antibiotics to every 100 mL of culture medium, which is also filtered through a 0.22 μm filter and stored at 4° C.

[0073] Specifically, the inducers in C2 included insulin, dexamethasone, IBMX, and a lipid mixture, with their concentrations reaching 20 μg / mL, 1 μmol / L, 10 nmol / L, and 100 μmol / L, respectively.

[0074] Example 1

[0075] The steps of the method for isolating and culturing grass carp preadipocytes are as follows:

[0076] (1) Preparation of cell culture reagents. The specific steps of cell culture reagents are as follows:

[0077] ① Grass carp preadipocyte culture medium: DMEM / F12 culture medium containing 10% fetal bovine serum, 3.57 mg / ml HEPES, 100 IU / mL penicillin and 100 μg / mL streptomycin.

[0078] ② Type I collagenase solution: 0.1 g type I collagenase and 2 g bovine serum albumin dissolved in 99 mL DMEM / F12 culture medium, 1 mL double antibody solution (10000 IU / mL penicillin, 10 mg / mL streptomycin).

[0079] ③PBS solution.

[0080] (2) Primary culture: The specific steps of primary culture are as follows:

[0081] ① Selection of experimental fish: Select healthy grass carp weighing 1-1.5kg.

[0082] ② Isolation of adipose tissue: The experimental fish were anesthetized with MS222 before dissection, and the gill arches were cut short to bleed; under sterile conditions, the surface of the experimental fish was wiped with 75% alcohol three times; the experimental fish were dissected with dissection tools. The peritoneal membrane of the experimental fish was cut open with sterile scissors, and the abdominal mesenteric adipose tissue was peeled off with forceps and sterile scissors.

[0083] ③ Cleaning of grass carp adipose tissue: Use ophthalmic forceps to peel off the peritoneal membrane remaining in the adipose tissue and remove obvious blood vessels; pick up the separated adipose tissue with forceps, rinse it in 70% alcohol for 15 seconds, then rinse it twice in a culture dish filled with PBS and 1% double antibody, and then rinse it once with DMEM / F12 culture medium.

[0084] ④ Enzymatic hydrolysis of grass carp adipose tissue: Place the washed adipose tissue in a culture dish containing 2-3 times the volume of type I collagenase and cut it into 1 mm pieces. 3 Transfer the chopped adipose tissue and collagenase into a 50ml centrifuge tube and place it on a shaker at 28 degrees for 30-60 minutes until there are no obvious tissue blocks, and terminate the digestion with a culture medium containing 10% fetal bovine serum.

[0085] ⑤ Isolation and culture of grass carp preadipocytes: After collagenase digestion, centrifuge at 2000rpm for 10min, discard the upper mature adipocytes and supernatant, add 2-3 times the volume of red blood cell lysis solution, lyse for 2min, neutralize with DMEM / F12 medium containing 10% fetal bovine serum, filter twice with a 200-mesh cell sieve, and centrifuge the obtained cell suspension at 3000rpm for 10min. Discard the supernatant, aspirate and discard the lower red blood cells, wash twice with fresh DMEM / F12 medium, resuspend the cells with preadipocyte medium, and culture in a 28℃ cell culture incubator with a 24-well cell culture plate. After 24 hours, change the medium and wash with PBS to remove non-adherent red blood cells and tissue blocks.

[0086] ⑥ Experimental results: On the third day of primary culture, a small amount of preadipocytes can be seen attached to the bottom of the plate ( Figure 2 ); When the cells grow to 80-90% contact, digestion and subculture are performed. On the third day after plating, cells can be seen to have converged to form a single cell layer. The cell characteristics are good and the growth state is good ( Figure 3 ), The light microscopic photograph of the third generation grass carp precursor fat cells shows that the cells are dividing vigorously and in good condition ( Figure 4 ).

[0087] Example 2

[0088] The steps of inducing grass carp preadipocytes are as follows: (based on Example 1)

[0089] (1) Grass carp preadipocyte induction medium:

[0090] The preadipocyte induction medium was DMEM / F12 medium containing 10 vol% fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine, 10 μg / ml insulin, 0.25 μM dexamethasone, 100 IU / mL penicillin and 100 μg / mL streptomycin.

[0091] (2) Induction of preadipocytes into mature adipocytes:

[0092] The cells were digested into single cells with 0.25% trypsin and cultured at 5 × 10 5 The cells were inoculated into 6-well plates at a concentration of 100 μg / well and cultured for 3 days, and then replaced with preadipogenic induction medium and cultured for 6 days. The cells were stained with Oil Red O and photographed under an inverted microscope.

[0093] The cells on the third day of isolation and culture were stained with Oil Red for observation. Figure 5 The results showed that the cells proliferated and divided, and the unilocular lipid droplet cells aggregated into clusters. Figure 6 The results of oil red staining of the second generation cells are shown in Table 1. Figure 7As shown, lipid droplets accumulated in the preadipocytes, the cell edges were blurred, and after the cells were stained with Oil Red O, red lipid droplets were present in the cells.

[0094] The beneficial effects of the present invention are:

[0095] The method for isolating and culturing grass carp precursor fat cells and inducing their differentiation simplifies the enzymatic hydrolysis method for primary culture of fish precursor tissue cells, combines tissue fragmentation with collagen enzymatic hydrolysis, and uses differential centrifugation to purify the precursor fat cells; by adjusting the formula of the culture medium, the pH stability of the culture medium can be better maintained; by adjusting the cell osmotic pressure, a large number of cell deaths due to the difference in internal and external osmotic pressures during digestion can be avoided.

[0096] The method for isolating and culturing grass carp preadipocytes and inducing their differentiation can adjust the pH value of the reaction system by adding HEPES to the preadipocyte culture medium, and can control a constant pH range for a long time, so as to effectively maintain the stability of cells and proteins, which is important for maintaining the stability of the internal and external environment of grass carp preadipocytes.

[0097] 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.

[0098] 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 isolating and culturing grass carp preadipocytes, characterized in that: The following steps are involved: S1. Under sterile conditions, grass carp adipose tissue was obtained and digested with type I collagenase digestion solution for 30-60 min to obtain grass carp preadipocytes; S2. The digested grass carp preadipocytes were inoculated into a 24-well cell plate, placed in a cell culture incubator at 28° C., and cultured using grass carp preadipocyte culture medium until the cells adhered to the wall.

2. The method for isolating and culturing grass carp preadipocytes according to claim 1, characterized in that: The specific method for obtaining grass carp adipose tissue in S1 is: A1. At room temperature, select a grass carp of 1-1.5 kg in size, wipe the surface of the fish with 75% alcohol, and dissect it in a sterile environment. A2. Take mesenteric fat tissue and soak it in 75% alcohol for 30 seconds; A3. Wash the fat three times with ice-cold PBS (pH 7.4) in a clean bench to remove connective tissue and blood as much as possible; A4. Wash the grass carp fat tissue blocks with DMEM / F12 culture medium.

3. The method for isolating and culturing grass carp preadipocytes according to claim 1, characterized in that: The specific way of digestion of grass carp adipose tissue in S1 is: B1. Place the washed adipose tissue in a culture dish containing 2-3 times the volume of type I collagenase digestion solution and cut it into pieces of about 1 mm3; B2. Transfer the chopped adipose tissue and collagenase digestion solution into a 50 ml centrifuge tube and place it on a shaker at 28°C for 30-60 minutes; B3. When there are no obvious tissue blocks in the centrifuge tube, terminate the digestion with culture medium containing 10% fetal bovine serum.

4. The method for isolating and culturing grass carp preadipocytes according to claim 1, characterized in that: The preparation method of the type I collagenase digestion solution in S1 is to add type I collagenase, bovine serum albumin, penicillin and streptomycin to DMEM / F12 culture medium containing calcium and magnesium ions; wherein the concentration of type I collagenase is 1 mg / ml, the concentration of bovine serum albumin is 2%, the concentration of penicillin is 100 IU / mL, and the concentration of streptomycin is 100 μg / mL.

5. The method for isolating and culturing grass carp preadipocytes according to claim 1, characterized in that: The specific steps in S1 are: S1-1. After the digestion of the culture medium is terminated, centrifuge at 2000 rpm for 10 min, discard the upper mature adipocytes and supernatant, add 2-3 times the volume of red blood cell lysis buffer, and lyse for 2 min; S1-2, after lysis, neutralize with DMEM / F12 medium containing 10% fetal bovine serum. After neutralization, filter twice with a 200-mesh cell sieve, and centrifuge the obtained cell suspension at 3000 rpm for 10 min. S1-3. After centrifugation, discard the supernatant and the lower layer of red blood cells, wash twice with fresh DMEM / F12 medium, resuspend the cells with growth medium DMEM / F12, 10% FBS, 1% double antibody, and culture in a 24-well cell culture plate; S1-4, after 24 hours of culture, the medium was changed and the cells were washed with PBS to remove non-adherent red blood cells and tissue blocks; S1-5. After two days, change the medium. When the cells proliferate to 80-90%, they can be digested and passaged.

6. The method for isolating and culturing grass carp preadipocytes according to claim 1, characterized in that: The culture medium for preadipocytes in S2 was DMEM / F12 medium containing 10 vol% fetal bovine serum, 3.57 mg / ml HEPES, 100 IU / mL penicillin and 100 μg / mL streptomycin.

7. A method for inducing differentiation of grass carp preadipocytes, using the method for isolating and culturing grass carp preadipocytes as claimed in claim 1, characterized in that: C1. Cell preparation: Grass carp preadipocytes are separated by the enzymatic hydrolysis method in S1, and the grass carp preadipocytes are cultured in a serum-containing medium to ensure their normal growth and proliferation; C2. Cell induction differentiation: When the grass carp preadipocytes grow to 80%-90% confluence in the culture dish, the induction differentiation is started; on the basis of the serum culture medium, an inducer is added to prepare an induction culture medium; the cell culture medium is replaced with the induction culture medium, and the induction differentiation process is started; C3. Differentiation process management: During the induction differentiation period, according to the cell growth and the color change of the culture medium, replace the fresh induction medium in time to ensure that the cells are in the best growth and differentiation environment; regularly observe the morphological changes of the cells and record the key time points in the differentiation process, such as the beginning of cell differentiation and the formation of fat droplets; C4. Completion of cell differentiation: After a period of induced differentiation, the degree of cell differentiation is evaluated by staining and microscopic observation; further experimental analysis is performed on the differentiated adipocytes according to the experimental purpose, such as detecting the expression levels of lipid metabolism-related genes and proteins, and studying the biological functions of adipocytes.

8. The method for inducing differentiation of grass carp preadipocytes according to claim 7, characterized in that: The serum-containing culture medium in C1 is prepared by adding 90 mL of DMEM / F12 culture medium, 10 mL of serum, 0.25 g of NaHCO 3 and 0.1 mL of antibiotics to every 100 mL of culture medium, and the medium is also filtered through a 0.22 μm filter and stored at 4° C.

9. The method for inducing differentiation of grass carp preadipocytes according to claim 7, characterized in that: The inducers in the C2 include insulin, dexamethasone, IBMX and lipid mixture, and their concentrations reach 20 μg / mL, 1 μmol / L, 10 nmol / L and 100 μmol / L, respectively.

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