CN115181721A - Method for inhibiting proliferation of subcutaneous fat cells and fat anabolism of mutton sheep - Google Patents
Method for inhibiting proliferation of subcutaneous fat cells and fat anabolism of mutton sheep Download PDFInfo
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- CN115181721A CN115181721A CN202210823317.XA CN202210823317A CN115181721A CN 115181721 A CN115181721 A CN 115181721A CN 202210823317 A CN202210823317 A CN 202210823317A CN 115181721 A CN115181721 A CN 115181721A
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- mutton sheep
- morin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0653—Adipocytes; Adipose tissue
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
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- A23K20/121—Heterocyclic compounds containing oxygen or sulfur as hetero atom
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
The invention provides a method for inhibiting proliferation of subcutaneous fat cells and fat anabolism of mutton sheep, belonging to the technical field of development of active substances of feed. The method comprises the following steps: inoculating the subcutaneous fat cells of the mutton sheep into a cell culture apparatus, and adding a cell culture medium; after the cells are attached to the wall, the morin is added into a cell culture medium for further cell proliferation culture or adipogenic culture. The method for culturing the subcutaneous fat cells of the mutton sheep can effectively reduce the proliferation of the subcutaneous fat cells of the mutton sheep and promote the apoptosis of the cells, and simultaneously, the method can effectively reduce the lipogenesis of the subcutaneous fat cells of the mutton sheep.
Description
Technical Field
The invention belongs to the technical field of feed active substance development, and particularly relates to a method for inhibiting subcutaneous fat cell proliferation and fat anabolism of mutton sheep.
Background
In mutton production, subcutaneous and intramuscular fat content in the carcass is an important index for assessing meat quality, wherein subcutaneous fat thickness affects sheep carcass quality and intramuscular fat content affects mutton quality. During the growth process of mutton sheep, the sequence of fat deposition is generally periorgan fat, subcutaneous fat and intramuscular fat. The fat formation of the animal body needs to take more energy substances, and the deposition of subcutaneous fat reduces the lean meat percentage of the mutton sheep carcass and increases the feeding cost of the mutton sheep. Therefore, effectively reducing the deposition of subcutaneous fat is one of the key points of cost saving and efficiency improvement in the mutton sheep industry.
The Morin (Morin) is a natural active substance separated from plants, is a secondary metabolite of polyphenols in the plants, and is widely distributed in the natural world. The molecular formula is C 15 H 10 O 7 The structure is that an oxygen-containing heterocyclic ring is connected with two aromatic rings. Research shows that the morin has good antioxidant and anticancer effects. Meanwhile, in vivo and in vitro experiments show that the morin has a remarkable anti-inflammatory effect. In some animal models, morin inhibits the expression of MMP-2 and MMP-9. In addition, the morin has the functions of resisting oxidation, reducing blood sugar and the like. It can be used for resisting viral infection, and treating gastropathy, chronic inflammation and coronary heart disease. Has antibacterial effect on typhoid bacillus, dysentery bacillus and Staphylococcus aureus. Therefore, the phellinus igniarius has wide market prospect and will have profound significance for development and utilization of the phellinus igniarius.
At present, the use of morin in the proliferation of subcutaneous fat cells and fat anabolism of sheep is not clear, and thus, the applicant has conducted studies thereon. The applicant finds that the morin serving as a natural polyphenol active substance in plants has the effects of inhibiting proliferation and lipid metabolism of subcutaneous fat cells of mutton sheep, and provides an effective method for inhibiting generation of subcutaneous fat, reducing accumulation of ineffective fat and improving production efficiency of the mutton sheep.
Disclosure of Invention
The invention aims to provide a method for inhibiting subcutaneous fat cell proliferation and fat anabolism of mutton sheep.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for inhibiting subcutaneous fat cell proliferation and fat anabolism in a meat sheep, comprising the steps of:
(1) Inoculating the subcutaneous fat cells of the mutton sheep into a cell culture apparatus, and adding a cell culture medium;
(2) After the cells are attached to the wall, the morin is added into a cell culture medium for further cell proliferation culture or adipogenic culture.
Application of morin in preparing biological agent for inhibiting proliferation of subcutaneous fat cells of mutton sheep.
Application of morin in preparing biological preparation for promoting apoptosis of subcutaneous fat cells of mutton sheep.
Application of morin in preparing a biological preparation for inhibiting the adipogenic process of subcutaneous fat cells of mutton sheep.
Application of the morin in preparing a biological preparation for promoting the expression of an apoptosis promoting gene Bax in subcutaneous fat cells of mutton sheep.
Application of morin in preparing a biological preparation for inhibiting expression of an apoptosis inhibiting gene Bcl-2 in subcutaneous fat cells of mutton sheep.
Application of morin in preparing a biological agent for promoting expression of a key lipolysis gene AMPK alpha in fat cells.
Application of morin in preparing biological agent for promoting gene expression of fatty triglyceride lipase and hormone sensitive lipase in fat cell.
Application of morin in preparing feed for inhibiting subcutaneous fat deposition of mutton sheep.
Preferably, the application comprises inhibiting proliferation of subcutaneous fat cells of the mutton sheep, promoting apoptosis of the subcutaneous fat cells of the mutton sheep and inhibiting lipogenesis of the subcutaneous fat cells of the mutton sheep.
The invention has the beneficial effects that:
the morin can effectively inhibit the proliferation of subcutaneous fat cells of the mutton sheep and promote the apoptosis of the subcutaneous fat cells of the mutton sheep;
meanwhile, the invention discovers that the morin can effectively inhibit fat anabolism of subcutaneous fat cells of the mutton sheep.
Drawings
FIG. 1 is a graph showing the effect of different concentrations of morin on apoptosis of subcutaneous fat cells of mutton sheep precursors;
FIG. 2 is a graph showing the effect of different concentrations of morin on adipogenesis in subcutaneous fat cells of mutton sheep.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1
Separation and purification of mutton sheep subcutaneous fat cells
1. Cleaning hair at sampling position of mutton sheep back, sterilizing with iodine tincture and alcohol, and surgery to obtain 1cm hair 3 Subcutaneous adipose tissue mass;
2. removing blood vessels and connective tissues visible in subcutaneous adipose tissues of mutton sheep in a super clean bench, washing for several times by PBS (phosphate buffer solution) containing 2% double antibodies (penicillin and streptomycin), cutting tissue blocks into small blocks while washing, and dipping PBS liquid on the surface of the tissue blocks for several times by using a disposable culture dish after washing till no water drops;
3. shearing the treated tissue block to be minced by using an operation scissors, attaching the tissue block to the bottom wall of a T25 cell culture bottle by using a pair of tweezers, turning over the culture bottle, slowly adding 5ml of cell culture solution (DMEM/F12 +10% FBS +1% double antibody), putting the culture bottle into an incubator to stand for 4h with the bottom wall upward, and slightly turning over the culture bottle to enable the culture solution to be in contact with the tissue block (if the tissue block floats, the turning time can be properly prolonged);
4. after the culture bottle is turned over and is subjected to static culture for 3 days, cells are free from the edge of the tissue block, and the culture bottle is prevented from shaking in the period so as to prevent the tissue block from floating. When the cells are paved to 30% of the bottom of the bottle, the liquid is changed slightly, adherent tissue blocks are kept as much as possible, then the liquid is changed timely according to the growth condition of the cells, and the cells can be digested and passaged when the cells grow to about 70%.
Example 2
Induction and differentiation of adipocytes
1. The subcutaneous fat cells of mutton sheep were diluted with DMEM/F12 growth medium (500 mL of DMEM/F12 medium +50mL of fetal bovine serum +5mL of diabody) and then treated with 6X 10 4 Per cm 2 Density inoculated into culture flask or culture plate, charged with 5% CO at 37 deg.C 2 Culturing in the incubator, and changing the growth culture solution every 2 days;
2. after the cells are converged, changing the growth culture solution into DMEM/F12 induced differentiation culture solution (DMEM/F12 growth culture solution +10 mu g/mL bovine insulin +0.5mM methyl isobutyl xanthine +1 mu M dexamethasone) to induce the precursor adipocytes to be differentiated into the adipocytes;
after 3.48h (differentiation was determined to be 0 d), the differentiation-inducing culture solution was replaced with a basal differentiation culture solution (DMEM/F12 growth medium + 10. Mu.g/mL bovine insulin).
Example 3
Determining the influence of different concentrations of morin on the proliferation of subcutaneous fat cells of mutton sheep
1. Will be 5X 10 4 Individual cell/cm 2 Density was seeded in 96-well plates and 5 wells were used as replicates.
After 2.24h, cells were treated with 0, 200 μ M,400 μ M morin (DMSO configuration);
3. after 48h of treatment, the supernatant was removed and 90. Mu.L of fetal calf serum free medium plus 10. Mu.L of CCK-8 solution was added to each well for 2 hours at 37 ℃;
4. the absorbance was measured at 450nm using a Biotek ELx808 microplate reader (Biotek) and the results are shown in Table 1.
TABLE 1 Effect of different concentrations of Phellinus on proliferation of subcutaneous adipocytes of mutton sheep precursors
From Table 1, it can be seen that the difference was statistically significant when 200. Mu.M of morin was added, the relative OD value was 0.94, when 400. Mu.M of morin was added, the OD value was 0.85, and the P value was less than 0.05. The results show that the addition of 200 mu M or 400 mu M of morin has a remarkable inhibition effect on proliferation of subcutaneous fat cells of mutton sheep precursors.
Example 4
Determining the influence of different concentrations of morin on the apoptosis of subcutaneous fat cells of mutton sheep
1.5×10 4 Individual cell/cm 2 Preparing cell climbing sheets in density;
2. cells were treated with 0, 200 μ M,400 μ M morin (DMSO configuration);
3. after 48h of treatment, the supernatant was removed and the cells were washed 2 times with phosphate buffer;
4. staining was performed using a Hoechst33342 live cell staining solution, and images of stained cells were taken by an inverted fluorescence microscope (Leica), and the results are shown in fig. 1.
As can be seen from the results of FIG. 1, the addition of morin to 400. Mu.M increased the intensity of nuclear staining, promoting the apoptosis process of subcutaneous adipocytes of mutton sheep precursors.
Example 5
Determination of the influence of different concentrations of Phellinus Linteus on apoptosis genes BAX and BCL-2 in subcutaneous fat cells of mutton sheep
1. Subcutaneous fat cells of mutton sheep in logarithmic growth phase were inoculated in 6-well culture plates, and after overnight culture, the cells were treated with 0, 200 μ M,400 μ M morin (DMSO configuration);
2. total RNA was extracted from each sample using TRIzol reagent (Invitrogen);
3. RNA purity and integrity was analyzed using a Nanodrop 2000 spectrometer (Thermo Scientific) by measuring the a260: a280 ratio (1.8-2.0) and electrophoresis in 1% agarose;
4. synthesizing cDNA using HiScript first strand cDNA Synthesis kit (Vazyme);
5. real-time fluorescent quantitative (q-PCR) primers for Bax and Bcl-2 were designed and synthesized with the following sequences:
6. q-PCR was performed using the ChamQ SYBR Color qPCR Master Mix (Vazyme) fluorescent dye and LightCycler (Roche) fluorescent quantifier under the conditions of stage 1, 95 ℃ for 30s; stage 2, 10s at 95 ℃ and 30s at 60 ℃,40 cycles; stage 3, 95 ℃ 15s,60 ℃ 60s and 95 ℃ 15s;
7. beta-actin is used as reference gene, 2 -ΔΔCT The method calculates the relative gene expression level and the obtained results are shown in table 2.
TABLE 2 Effect of different concentrations of Phellinus on the relative expression level of apoptosis-related genes
As can be seen from Table 2, after the cells are treated by the morin, the relative expression level of the gene Bax which reflects the pro-apoptosis process is up-regulated, and the relative expression level of the gene Bcl-2 which inhibits the apoptosis process is down-regulated, which indicates that the addition of the morin promotes the apoptosis process of the subcutaneous fat cells of the mutton sheep.
Example 6
Determination of the Effect of different concentrations of Phellinus on fat production in subcutaneous fat cells of mutton sheep
1. Fat-forming differentiated mutton sheep fat cells are divided into 5 multiplied by 10 4 Individual cell/cm 2 Density plated on coverslips in 6-well plates, incubated overnight with 2ml growth medium;
2. cells were treated with 0, 200 μ M,400 μ M morin (DMSO configuration);
3. after 48h of treatment, cells were stained using an oil red O staining kit;
4. after staining, stained cells were photographed using a fluorescence microscope, and the results obtained are shown in fig. 2.
As can be seen from FIG. 2, the number of lipid droplets in adipocytes was significantly reduced after the addition of morin, indicating that the addition of morin inhibited the fat production process of mutton sheep differentiating subcutaneous adipocytes.
Example 7
1. Subcutaneous fat cells of mutton sheep in logarithmic growth phase were inoculated in 6-well culture plates, and after overnight culture, the cells were treated with 0, 200 μ M,400 μ M morin (DMSO configuration);
2. total RNA was extracted from each sample using TRIzol reagent (Invitrogen);
3. RNA purity and integrity was analyzed by measuring a260: a280 ratio (1.8-2.0) and electrophoresis in 1% agarose using a Nanodrop 2000 spectrometer (Thermo Scientific);
4. synthesizing cDNA using HiScript first strand cDNA Synthesis kit (Vazyme);
5. real-time fluorescent quantitative (q-PCR) primers for AMPK alpha, ATGL and HSL were designed and synthesized, with the following sequences:
6. q-PCR was performed using the ChamQ SYBR Color qPCR Master Mix (Vazyme) fluorescent dye and LightCycler (Roche) fluorescent quantifier under the conditions of stage 1, 95 ℃ for 30s; stage 2, 10s at 95 ℃ and 30s at 60 ℃,40 cycles; stage 3, 95 ℃ 15s,60 ℃ 60s and 95 ℃ 15s;
7. beta-actin is used as reference gene, 2 -ΔΔCT The relative gene expression levels were calculated by the method, and the obtained results are shown in Table 3.
TABLE 3 influence of different concentrations of Phellinus linteus flavin on relative expression level of genes involved in lipid metabolism
As can be seen from Table 3, after the addition of the morin, the key gene AMPKa for promoting lipolysis in subcutaneous fat cells of the mutton sheep is up-regulated, and the relative expression levels of the genes of lipolysis-related enzymes, namely, the triglyceride lipase (ATGL) and the Hormone Sensitive Lipase (HSL), are up-regulated. The fact that the morin can inhibit fat anabolism of subcutaneous fat cells of the mutton sheep is shown.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (9)
1. A method for inhibiting proliferation of subcutaneous fat cells and fat anabolism in mutton sheep, comprising the steps of:
(1) Inoculating the subcutaneous fat cells of the mutton sheep into a cell culture apparatus, and adding a cell culture medium;
(2) After the cells are attached to the wall, the morin is added into a cell culture medium for further cell proliferation culture or adipogenic culture.
2. Application of morin in preparing biological agent for inhibiting proliferation of subcutaneous fat cells of mutton sheep.
3. Application of morin in preparing biological agent for promoting apoptosis of subcutaneous fat cells of mutton sheep.
4. Application of morin in preparing a biological preparation for inhibiting the adipogenic process of subcutaneous fat cells of mutton sheep.
5. Application of the morin in preparing a biological preparation for promoting the expression of an apoptosis promoting gene Bax in subcutaneous fat cells of mutton sheep.
6. Application of morin in preparing a biological preparation for inhibiting expression of an apoptosis inhibiting gene Bcl-2 in subcutaneous fat cells of mutton sheep.
7. Application of morin in preparing a biological agent for promoting expression of a key gene AMPK alpha of lipolysis in fat cells.
8. Application of morin in preparing biological agent for promoting gene expression of fatty triglyceride lipase and hormone sensitive lipase in fat cell.
9. Application of morin in preparing feed for inhibiting subcutaneous fat deposition of mutton sheep.
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