Method for improving lipid droplet generation in bovine adipocyte differentiation process and promoting differentiation process

Abstract

The invention discloses a method for improving lipid droplet generation and promoting the differentiation process in the bovine adipocyte differentiation process, which comprises the following steps: (1) recovering primary bovine adipocytes, and culturing in a complete growth culture medium; (2) performing induced differentiation on the bovine adipocytes in an induced differentiation culture medium; and (3) replacing the bovine adipocytes subjected to induced differentiation with a culture medium containing 50-100 [mu] mol / L of linoleic acid, and culturing the bovine adipocytes. It is found for the first time that after bovine adipocyte differentiation is finished, culture continues to be conducted in a culture medium added with LA, lipid droplet generation in the bovine adipocyte differentiation process is improved, and the differentiation process is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a method for increasing lipid droplet generation during the differentiation of bovine adipocytes and promoting the differentiation process. Background Art

[0002] Unsaturated fatty acids are divided into monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs). PUFAs are straight-chain fatty acids with two or more double bonds and a carbon chain length of 18-22 carbon atoms. In PUFAs, the first unsaturated double bond is located between the 3rd and 4th carbon atoms from the methyl end, called -3 (n-3) PUFAs, and between the 6th and 7th carbon atoms, called -6 (n-6) PUFAs. Polyunsaturated fatty acids are important components of cell membranes and have a variety of physiological effects on maintaining normal body health. They not only provide essential fatty acids for the normal growth of the body, but also play an important physiological function in regulating bone development, fat metabolism, inhibiting the production of inflammatory factors, improving the body's immune function, and regulating intestinal health. n-6 PUFAs include linoleic acid (LA), arachidonic acid (AA), etc.

[0003] The role of polyunsaturated fatty acids in a variety of metabolic diseases has been widely confirmed. However, the role of different dietary n-6 / n-3PUFAs composition ratios in a variety of metabolic diseases such as cardiovascular disease and type 2 diabetes is still controversial. Studies have found that when rats were fed n-6 / n-3PUFA 10:1 and 20:1 groups for 12 weeks, blood glucose, serum total cholesterol (TC), and triglyceride (TG) in the experimental group were significantly higher than those in the control group. Changes in the type, content, and ratio of polyunsaturated fatty acids in feed will cause a decline in poultry growth and immune function, and will also cause a series of problems such as excessive deposition of poultry body fat. However, there is still much controversy about the role of n-6PUFAs in the differentiation of adipocytes. Some studies have found that 100umol / L LA induced a significant increase in the expression of PPARγmRNA in 3T3-L1 cell lines, but some studies have also found that n-3 and n-6PUFAs are inhibitors of SREBP-1, and PUFAs can inhibit SREBP-1c to regulate the expression of FASN and ACC, thereby inhibiting the differentiation of adipocytes. Therefore, its effect on adipocyte differentiation is not clear, and the regulatory mechanism is not yet clear.

[0004] In addition, the expression of transcription factors during adipocyte differentiation varies between species, and the expression patterns of proteins of the same family in different animals during lipid deposition also differ. In large livestock cattle, no detailed research reports have been reported on the regulatory mechanism of polyunsaturated fatty acid supplementation on growth and fattening or adipocyte differentiation, and this research is still blank. Summary of the invention

[0005] Purpose of the invention: The present invention aims to provide a method for increasing the generation of lipid droplets during the differentiation of bovine adipocytes and promoting the differentiation process.

[0006] Technical solution: The method of increasing the generation of lipid droplets during the differentiation of bovine adipocytes and promoting the differentiation process of the present invention comprises the following steps:

[0007] (1) reviving primary bovine adipocytes and culturing them in complete growth medium;

[0008] (2) replacing the bovine adipocytes with differentiation induction medium to induce differentiation;

[0009] (3) The induced differentiated bovine adipocytes were cultured in a culture medium containing 50-100 μmol / L linoleic acid.

[0010] Preferably, the bovine adipocytes are bovine intramuscular adipocytes.

[0011] Preferably, in step (1), the complete growth medium is based on Dulbecco's modified Eagle medium supplemented with 15% fetal bovine serum and 1% penicillin / streptomycin. More preferably, Dulbecco's modified Eagle medium (DMEM) / F-12 (DMEM / F-12, Gibco) is used as the basal medium.

[0012] Preferably, in step (1), the culture is as follows: after the bovine adipocytes are revived, when the culture density reaches 70%, the culture medium is switched to an induction medium to avoid spontaneous differentiation.

[0013] Preferably, in step (2), the differentiation induction medium is Dulbecco's modified Eagle medium supplemented with 0.5 mM isobutylmethylxanthine (IBMX), 1 mg / ml insulin, 1 μM dexamethasone (Dex), 15% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin. More preferably, Dulbecco's modified Eagle medium (DMEM) is used as DMEM / F-12 (DMEM / F-12, Gibco).

[0014] Preferably, in step (2), the time for inducing differentiation is 48 hours. The time of adding IBMX, Insulin and Dex is defined as the start of inducing differentiation, that is, day 0.

[0015] Further preferably, in step (3), the concentration of linoleic acid is 75-100 μmol / L.

[0016] Preferably, in step (3), the culture medium is Dulbecco's modified Eagle medium supplemented with 15% fetal bovine serum and linoleic acid solution, and more preferably Dulbecco's modified Eagle medium (DMEM) / F-12 (DMEM / F-12, Gibco).

[0017] Preferably, the linoleic acid solution is prepared by dissolving linoleic acid in anhydrous ethanol and then adding the linoleic acid into a 0.1 N NaOH solution.

[0018] Taking the culture medium containing 100 μmol / L LA as an example, the preparation method is: prepare 0.1N NaOH solution, dissolve 25 mg of LA in 2 mL of anhydrous ethanol, calculate its concentration to be 0.178 mmol / mL, take 280 μL LA and dissolve it in 1 ml of 0.1N NaOH solution, and dissolve LA in 15% BSA solution at a molar ratio of 1:1.

[0019] Preferably, in step (3), the cell culture medium is replaced with new one every 48 hours during the culture process, and the culture is carried out for a total of 144 hours.

[0020] After the cultivation technology in step (3), RNA extraction, reverse transcription and protein extraction were performed on the bovine intramuscular adipocytes. The content of metabolites related to lipid deposition in bovine intramuscular was detected using a lipid metabolite detection kit. The mRNA and protein expression changes of key genes PLIN1 and FASN related to early lipid droplet formation and lipid synthesis in adipocytes were further systematically detected by qRT-PCR technology and Western blot experiments.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention first discovered that after the differentiation of bovine adipocytes is completed, continuing to culture in a culture medium supplemented with LA improves the generation of lipid droplets during the differentiation of bovine adipocytes and promotes the differentiation process; (2) Compared with the culture method containing 100 nM insulin in the prior art, the addition of LA at a concentration of 100 μmol / L can significantly improve the degree of differentiation of bovine adipocytes and the formation of lipid droplets. On this basis, it also provides a certain experimental basis for solving the problem of slow lipid deposition in cattle. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1BODIPY staining of lipid droplets in bovine adipocytes after treatment with medium containing different concentrations of LA;

[0023] Figure 2 The mRNA and protein expression of key genes related to lipid synthesis in the 100 μmol / L LA-added group and the control group (** represents P<0.01, the difference is extremely significant). DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with embodiments.

[0025] The sources of cells and reagents involved in the examples are as follows:

[0026] (1) Primary bovine intramuscular adipocytes were isolated and preserved by the National Beef Cattle Improvement Center Laboratory;

[0027] (2) Insulin, dexamethasone, and 3-isobutyl-1-methylxanthine were purchased from Invitrogen;

[0028] (3) High-fidelity DNA polymerase Q5, purchased from NEB (Massachusetts, USA);

[0029] (4) RNA extraction reagent TRIzol, purchased from Solarbio;

[0030] (5) PrimerScript RT Master Mix reverse transcription kit and SYBR Premix EX Taq real-time fluorescence quantitative PCR kit were purchased from Takara;

[0031] (6) Western blot test related reagents were purchased from Solarbio;

[0032] (7) For detailed procedures of routine cell biology and molecular biology experiments such as cell culture, induced differentiation, RNA extraction, and protein extraction, please refer to Cell Culture Technology and Molecular Cloning (3rd Edition).

[0033] (8) Fetal bovine serum was purchased from Invitrogen, DMEM culture medium and double antibody were purchased from Hyclone, and transfection reagent was purchased from Promega.

[0034] Example 1: Cell recovery and culture

[0035] The isolated bovine intramuscular adipocytes were revived and cultured in complete growth medium containing Dulbecco's modified Eagle's medium / F-12 (DMEM / F-12, Gibco), 15% fetal bovine serum (Gibco) and 1% penicillin / streptomycin under 5% CO2 , 37°C. The growth medium was changed every two days and the cells were passaged at 70% confluence to avoid spontaneous differentiation.

[0036] Example 2: Cell differentiation induction

[0037] According to the classic induction scheme, the adipocyte differentiation induction medium was prepared with isobutylmethylxanthine (IBMX, 0.5mM), insulin (Insulin, 1mg / ml) and dexamethasone (Dex 1μM) 15% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin culture medium (DMEM / F-12). When the cell density reached 70%, the bovine intramuscular adipocytes were induced to differentiate, and the differentiation induction culture was carried out for 48 hours. Induction differentiation was defined as day 0.

[0038] Example 3: Screening for suitable LA addition concentration

[0039] Step 1: Preparation of culture medium solution

[0040] Prepare 0.1N NaOH solution, dissolve 25mg of LA in 2mL of anhydrous ethanol, calculate its concentration to be 0.178mmol / mL, take 280μL of LA and dissolve it in 1ml of 0.1N NaOH solution, dissolve LA in 15% BSA solution (i.e. F-12DMEM supplemented with 15% fetal bovine serum) at a molar ratio of 1:1, and obtain a culture medium containing LA at a concentration of 100μmol / L. According to this method, prepare culture medium containing LA at concentrations of 0μmol / L, 25μmol / L, 50μmol / L, 75μmol / L, 100μmol / L, 200μmol / L, and 400μmol / L.

[0041] (2) The induced differentiated bovine intramuscular adipocytes were cultured in a culture medium containing LA at concentrations of 0 μmol / L, 25 μmol / L, 50 μmol / L, 75 μmol / L, 100 μmol / L, 200 μmol / L, and 400 μmol / L (culture conditions: 5% CO 2 , 37°C), and the culture medium was replaced every 48 hours for a total of 144 hours. Each test group was repeated three times, with 6 wells in each group.

[0042] Example 4: Detection of changes in lipid droplets during differentiation of bovine intramuscular adipocytes

[0043] After being treated with medium containing different concentrations of LA for 144 hours, BODIPY lipid droplet staining was used to detect the changes in lipid droplets during the differentiation of bovine intramuscular adipocytes.

[0044] BODIPY lipid droplet staining: The differentiated bovine intramuscular adipocyte test group and the treatment group with a confluence of 95% were washed with 1×PBS for 3 times (5 min / time), and 4% paraformaldehyde was added to the cells in the culture dish for fixation for 1 hour to avoid shaking. Continue to wash the cells in the culture dish 3 times (5 min / time) with 1×PBS. Dissolve BODIPY in DMSO and dilute it with PBS (1:1000), let it stand at room temperature, and stain for 30 minutes. Wash three times with 1×PBS, and finally examine under a fluorescence microscope. The results are as follows Figure 1 shown.

[0045] Depend on Figure 1 The results of BODIPY lipid droplet staining showed that compared with the control group, the number of lipid droplets increased when 50-100 μmol / L LA was added, and the number of lipid droplets in the 100 μmol / L LA addition group was the largest compared with other groups. This result shows that the addition of 50-100 μmol / L LA promotes the generation and differentiation of lipid droplets during the differentiation of bovine intramuscular adipocytes. In addition, when 200 μmol / L LA and above were added, some cells shrank and their morphology changed.

[0046] Example 5: Detection of mRNA and protein expression of key genes

[0047] The qRT-PCR technology and Western blot experimental system were used to detect the changes in the mRNA and protein levels of key genes PLIN1 and FASN related to early lipid droplet formation and lipid synthesis in adipocytes when 100 μmol / L LA was added.

[0048] The changes of lipid synthesis-related genes (PLIN1, FASN) were detected by real-time fluorescence quantitative PCR and other experimental methods. The primer sequences were designed using primer5.0 software according to the bovine PLIN1 and FASN gene sequences on NCBI (see Table 1 for details) and sent to Shanghai Biosynthetics.

[0049] Primer sequences:

[0050] Table 1 Real-time fluorescence quantitative primer sequences

[0051]

[0052] Real-time fluorescence quantitative PCR: The TRIzol method was used to extract RNA from primary bovine intramuscular adipocytes, and the PrimerScriptRTMaster Mix reverse transcription kit and the corresponding program were used for reverse transcription to obtain bovine adipocyte cDNA. Real-time fluorescence quantitative PCR used cDNA as a template and used NEB's Q5 high-fidelity DNA polymerase to PCR amplify PLIN1 and FASN genes, with the internal reference gene being the β-actin gene. The PCR amplification reaction system was: Q5 High-Fidelity 2X Master Mix 12.5μL, Forward Primer 1.5μL, Reverse Primer 1.5μL, Template DNA 100ng, and ddH 2 Reaction conditions: Real-time fluorescence quantitative PCR reaction system 20 μL: SYBR Premix Ex TaqⅡ 10 μL, upstream and downstream primers 0.8 μL each, ROX Reference Dye (50×) 0.4 μL, template DNA 2 μL, RNase-free ddH 2 O 6μL. PCR reaction program: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 60℃ annealing for 34s, 40 cycles. Each sample was repeated 3 times.

[0053] The Western blot detection method for the target protein is to add PMSF to the protein extract at a ratio of 1:100 to obtain a protein lysate, and let it stand on ice for 20 minutes. Use a cell scraper to collect the fully lysed cell suspension in a 1.5mL centrifuge tube and freeze it at 14000g for 10 minutes. Aspirate the supernatant, add 40μL protein loading buffer to 160μL supernatant to obtain the prepared protein sample, boil it in a water bath for 10 minutes and then divide it. Prepare 10% separation gel and 5% concentration gel, and load the protein sample for electrophoresis. After electrophoresis, transfer the membrane, and then block it with membrane blocking solution for 30 minutes. After blocking, place the membrane in a clean box, take 5mL TBST each time to clean the residual blocking solution on the membrane surface, and wash it 3 times in total, each time for 10 minutes. After washing, the antibody was incubated at 4°C overnight. The primary antibodies were Anti-PLIN1 (1:2500) and Anti-FASN. The expression of the target protein was detected with β-actin protein as the internal reference. Anti-β-actin (1:5000) was diluted and incubated at 4°C overnight. 5 mL of TBST was taken to wash the membrane surface each time. The washing was performed 3 times, each time for 10 minutes. The secondary antibody goat anti-rabbit anti-IgG H&L (HRP) (1:5000) was diluted with antibody diluent and incubated on a shaker at room temperature for 2 hours. 5 mL of TBST was taken to wash the membrane surface each time. After 3 times, the substrate chemiluminescence and development were performed to detect the expression of the target protein.

[0054] Test results such as Figure 2 shown.

[0055] Depend on Figure 2 It can be seen that the mRNA and protein expressions of the key genes for lipid synthesis, PLIN1 and FASN, were higher than those in the control group. PLIN1 protein is a protein that can be anchored on the surface of lipid droplets during the formation of lipid droplets. Its high expression level reflects the large number of lipid droplets generated under this condition; FASN is a key enzyme in the process of fatty acid synthesis during adipocyte differentiation. Its high expression level indicates a high degree of lipid synthesis. In summary, it is shown that the addition of 100 μmol / L LA promotes the formation of lipid droplets during the differentiation of bovine intramuscular adipocytes and promotes the lipid synthesis process.

Claims

1. A method for increasing lipid droplet generation and promoting differentiation during bovine adipocyte differentiation, characterized in that: The following steps are involved: (1) reviving primary bovine adipocytes and culturing them in complete growth medium; (2) replacing the bovine adipocytes with differentiation induction medium to induce differentiation; (3) The induced differentiated bovine adipocytes were cultured in a culture medium containing 50-100 μmol / L linoleic acid.

2. The method for increasing lipid droplet generation and promoting differentiation during bovine adipocyte differentiation according to claim 1, characterized in that: The bovine adipocytes are bovine intramuscular adipocytes.

3. The method for increasing lipid droplet generation and promoting differentiation during bovine adipocyte differentiation according to claim 1, characterized in that: In step (3), the concentration of linoleic acid is 75-100 μmol / L.

4. The method for increasing lipid droplet generation and promoting differentiation during bovine adipocyte differentiation according to claim 1, characterized in that: In step (3), the culture medium is based on Dulbecco's modified Eagle medium, supplemented with 15% fetal bovine serum and linoleic acid solution.

5. The method for increasing lipid droplet generation and promoting differentiation during bovine adipocyte differentiation according to claim 4, characterized in that: The method for preparing the linoleic acid solution is as follows: dissolving linoleic acid in anhydrous ethanol, and then adding the linoleic acid into a 0.1 N NaOH solution.

6. The method for increasing lipid droplet generation and promoting differentiation during bovine adipocyte differentiation according to claim 1, characterized in that: In step (3), the cell culture medium is replaced with new one every 48 hours during the culture process, and the culture is carried out for a total of 144 hours.

7. The method for increasing lipid droplet generation and promoting differentiation during bovine adipocyte differentiation according to claim 1, characterized in that: In step (1), the complete growth medium is based on Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum and 1% penicillin / streptomycin.

8. The method for increasing lipid droplet generation and promoting differentiation during bovine adipocyte differentiation according to claim 1, characterized in that: In step (1), the culture is as follows: after the bovine fat cells are revived, when the culture density reaches 70%, the induction culture medium is replaced.

9. The method for increasing lipid droplet generation and promoting differentiation during bovine adipocyte differentiation according to claim 1, characterized in that: In step (2), the differentiation induction medium is Dulbecco's modified Eagle's medium supplemented with 0.5 mM isobutylmethylxanthine, 1 mg / ml insulin, 1 μM dexamethasone, 15% fetal bovine serum, 1% penicillin and 1% streptomycin.

10. The method for increasing lipid droplet generation and promoting differentiation during bovine adipocyte differentiation according to claim 1, characterized in that: In step (2), the differentiation induction time is 48 hours.

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