CN115039841B - Promoter for promoting differentiation of porcine skeletal muscle satellite cells - Google Patents
Promoter for promoting differentiation of porcine skeletal muscle satellite cells Download PDFInfo
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- CN115039841B CN115039841B CN202210804681.1A CN202210804681A CN115039841B CN 115039841 B CN115039841 B CN 115039841B CN 202210804681 A CN202210804681 A CN 202210804681A CN 115039841 B CN115039841 B CN 115039841B
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
The invention provides an accelerator for promoting porcine skeletal muscle satellite cell differentiation, belonging to the technical field of porcine development. The promoter is the stephania polysaccharide, and the stephania polysaccharide can obviously promote the protein expression of the proteins MyHC and MyoG related to the differentiation of the skeletal muscle satellite cells of pigs through Western Blot detection. Therefore, the stephania polysaccharide can be used for preparing the porcine skeletal muscle satellite cell differentiation promoter.
Description
The scheme is a divisional application, and the name of the original application is: the application of the stephania root extract in preparing feed for promoting the development of pig muscle is as follows: 2020-09-02, the application number of the original application is: CN202010907868.5.
Technical Field
The invention belongs to the technical field of pig development, and particularly relates to an accelerator for promoting differentiation of skeletal muscle satellite cells of pigs.
Background
Pork is the most edible meat in our country and is one of the most economical animal protein sources in our country. Compared with other livestock, the pig has the advantages of high reproductive capacity and high growth speed. Currently, the pig industry is already the pillar industry in our agriculture and animal husbandry. Along with the development of the economic level of China and the improvement of the living standard of people, the requirements of consumers on pork quality are higher and higher. Meat quality has been an important economic indicator, wherein muscle fiber is a fundamental unit constituting pig muscle, and is closely related to the quality of meat produced by pigs. The myofiber is formed by differentiation and fusion of skeletal muscle satellite cells, so that the research on the mechanism of the growth and development process of the skeletal muscle satellite cells of pigs has important significance for improving the meat productivity of live pigs, improving the quality of pork and meeting the requirements of people on pork.
Disclosure of Invention
The invention aims to provide an application of stephania cepharantha polysaccharide in promoting proliferation and cell differentiation of porcine skeletal muscle satellite cells.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention provides an application of stephania polysaccharide in preparing feed for promoting pig muscle development.
Preferably, the preparation method of the stephania polysaccharide comprises the following steps:
(1) Cleaning semen Euphorbiae, oven drying, and pulverizing into powder to obtain semen Euphorbiae powder;
(2) Adding 20 times of petroleum ether-methanol mixed solution (2:1), degreasing for 5h to obtain degreased stephania japonica mixed solution;
(3) Filtering the defatted stephania japonica mixed solution to obtain stephania japonica filter residues;
(4) Adding 25 times of water into the euphorbia lathyris filter residue, and leaching for 5 hours at 85 ℃ to obtain euphorbia lathyris filter liquor;
(5) Filtering with ultrafiltration membrane with molecular weight cutoff of 10000 to remove impurities, and concentrating the semen Euphorbiae filtrate under reduced pressure to 1/3 of the original volume to obtain semen Euphorbiae Pulveratum Jin Zicu polysaccharide solution;
(6) Adding 4 times of 95% ethanol, precipitating with ethanol for 24h, centrifuging at 8000rpm/min for 10min, and removing supernatant to obtain thousands of Jin Zicu polysaccharides;
(7) Washing the Qian Jin Zicu polysaccharide with absolute ethanol and acetone, removing protein by sevage method, and freeze drying to obtain Qian jin Zi polysaccharide.
In addition, the invention provides application of stephania sinica diels polysaccharide in preparing a pig skeletal muscle satellite cell proliferation promoting agent, and the stephania sinica diels polysaccharide is prepared by the following steps:
(1) Cleaning semen Euphorbiae, oven drying, and pulverizing into powder to obtain semen Euphorbiae powder;
(2) Adding 20 times of petroleum ether-methanol mixed solution (2:1), degreasing for 5h to obtain degreased stephania japonica mixed solution;
(3) Filtering the defatted stephania japonica mixed solution to obtain stephania japonica filter residues;
(4) Adding 25 times of water into the euphorbia lathyris filter residue, and leaching for 5 hours at 85 ℃ to obtain euphorbia lathyris filter liquor;
(5) Filtering with ultrafiltration membrane with molecular weight cutoff of 10000 to remove impurities, and concentrating the semen Euphorbiae filtrate under reduced pressure to 1/3 of the original volume to obtain semen Euphorbiae Pulveratum Jin Zicu polysaccharide solution;
(6) Adding 4 times of 95% ethanol, precipitating with ethanol for 24h, centrifuging at 8000rpm/min for 10min, and removing supernatant to obtain thousands of Jin Zicu polysaccharides;
(7) Washing the Qian Jin Zicu polysaccharide with absolute ethanol and acetone, removing protein by sevage method, and freeze drying to obtain Qian jin Zi polysaccharide.
In addition, the invention provides application of stephania sinica Diels polysaccharide in preparing a pig skeletal muscle satellite cell differentiation promoter, and the preparation method of the stephania sinica Diels polysaccharide comprises the following steps:
(1) Cleaning semen Euphorbiae, oven drying, and pulverizing into powder to obtain semen Euphorbiae powder;
(2) Adding 20 times of petroleum ether-methanol mixed solution (2:1), degreasing for 5h to obtain degreased stephania japonica mixed solution;
(3) Filtering the defatted stephania japonica mixed solution to obtain stephania japonica filter residues;
(4) Adding 25 times of water into the euphorbia lathyris filter residue, and leaching for 5 hours at 85 ℃ to obtain euphorbia lathyris filter liquor;
(5) Filtering with ultrafiltration membrane with molecular weight cutoff of 10000 to remove impurities, and concentrating the semen Euphorbiae filtrate under reduced pressure to 1/3 of the original volume to obtain semen Euphorbiae Pulveratum Jin Zicu polysaccharide solution;
(6) Adding 4 times of 95% ethanol, precipitating with ethanol for 24h, centrifuging at 8000rpm/min for 10min, and removing supernatant to obtain thousands of Jin Zicu polysaccharides;
(7) Washing the Qian Jin Zicu polysaccharide with absolute ethanol and acetone, removing protein by sevage method, and freeze drying to obtain Qian jin Zi polysaccharide.
The invention has the beneficial effects that:
the invention discovers that the stephania sinica Diels polysaccharide can effectively promote proliferation and differentiation of porcine skeletal muscle satellite cells, so that the stephania sinica Diels polysaccharide can be used for preparing porcine skeletal muscle satellite cell proliferation and differentiation promoters, and the stephania sinica Diels polysaccharide can be used for preparing feed for promoting porcine muscle development.
Drawings
FIG. 1 EDU staining results
FIG. 2 MyHC and MyoG Western Blot test results.
Detailed Description
Example 1
(1) Cleaning semen Euphorbiae, oven drying, and pulverizing into powder to obtain semen Euphorbiae powder;
(2) Adding 20 times of petroleum ether-methanol mixed solution (2:1), degreasing for 5h to obtain degreased stephania japonica mixed solution;
(3) Filtering the defatted stephania japonica mixed solution to obtain stephania japonica filter residues;
(4) Adding 25 times of water into the euphorbia lathyris filter residue, and leaching for 5 hours at 85 ℃ to obtain euphorbia lathyris filter liquor;
(5) Filtering with ultrafiltration membrane with molecular weight cutoff of 10000 to remove impurities, and concentrating the semen Euphorbiae filtrate under reduced pressure to 1/3 of the original volume to obtain semen Euphorbiae Pulveratum Jin Zicu polysaccharide solution;
(6) Adding 4 times of 95% ethanol, precipitating with ethanol for 24h, centrifuging at 8000rpm/min for 10min, and removing supernatant to obtain thousands of Jin Zicu polysaccharides;
(7) Washing the Qian Jin Zicu polysaccharide with absolute ethanol and acetone, removing protein by sevage method, and freeze drying to obtain Qian jin Zi polysaccharide.
Isolation of porcine skeletal muscle satellite cells
1. After the 7-day-old large white piglets are killed, all muscles of the hind legs of the piglets are taken and placed in 30ml PBS containing double antibodies, and after rinsing, fascia on the surfaces of the muscles are removed and the muscle tissues are sheared into 1mm 3 Is a meat emulsion;
2. transferring the minced meat into a centrifuge tube, centrifuging at 1000rpm/min for 10min, transferring the precipitate into a culture dish, adding 0.1% type II collagenase, and placing in a 37 ℃ incubator for digestion for 1-2h;
3. after digestion, filtering the suspension by using a cell sieve with the diameter of 100 mu m, transferring the obtained filtrate into a new centrifuge tube, centrifuging at 2000rpm/min for 10min;
4. removing the supernatant, re-suspending cells by PBS, centrifuging at 1500rpm/min for 10min;
5. removing the supernatant, adding erythrocyte lysate to resuspend, standing for 10min, filtering the suspension by using a 40 mu m cell sieve, transferring the liquid into a new centrifuge tube, centrifuging at 1500rpm/min for 10min;
6. the supernatant was removed, the cells were resuspended in the complete medium of the experiment, inoculated into a petri dish, and placed in a cell incubator for culture.
Example 3
Stephania sinica Diels polysaccharide for promoting proliferation of skeletal muscle satellite cells of pigs
(1) Inoculating 2000 pig skeletal muscle satellite cells in logarithmic growth phase into 96-well plate, culturing overnight, and co-setting;
(2) Changing to complete culture medium containing 0mg/ml semen Euphorbiae polysaccharide, 25mg/ml semen Euphorbiae polysaccharide, 50mg/ml semen Euphorbiae polysaccharide, 100mg/ml semen Euphorbiae polysaccharide, 200mg/ml semen Euphorbiae polysaccharide, culturing for 48 hr, adding 10 μl CCK-8, and detecting 450nm OD value.
The experimental results are shown in table 1:
TABLE 1 Effect of varying concentrations of Stephania sinica Diels polysaccharide on porcine skeletal muscle satellite cell proliferation
| Additives | 0mg/ml stephania sinica Diels polysaccharide | 25mg/ml stephania sinica Diels polysaccharide | 50mg/ml stephania sinica Diels polysaccharide | 100mg/ml stephania sinica Diels polysaccharide | 200mg/ml stephania sinica Diels polysaccharide |
| OD value | 0.594±0.031 | 0.644±0.021 | 0.732±0.026 | 0.810±0.022 | 0.907±0.014 |
From the table, the stephania polysaccharide can significantly promote the cell proliferation of the porcine skeletal muscle satellite cells.
Example 4
Edu staining further demonstrates the effect of Stephania cepharantha polysaccharide on cell proliferation of porcine skeletal muscle satellite cells
(1) Pig skeletal muscle satellite cells are inoculated into a 96-well plate, treated with 0mg/ml stephania cepharantha polysaccharide and 200mg/ml stephania cepharantha polysaccharide for 24 hours, the culture medium is removed, and 100 μl EDU is added for 2 hours;
(2) EDU was removed and cells were washed 2 times with PBS for 5min each;
(3) Adding 4% paraformaldehyde, fixing at room temperature for 30min, removing the fixing solution, adding 100 μl of 0.2% glycine, incubating for 5min, and washing the cells with PBS for 2 times each for 5min;
(4) 100 μl of 0.5% Triton-100 was added and treated for 10min and washed with PBS for 5min;
(5) 100 μl of Apollo was added to each well and treated for 10min, and washed with PBS for 5min;
(6) Adding 100 μl of 0.5% Triton-100 shaking table for 2 times each for 10min;
(7) Adding 100 μl of methanol for 2 times, each time 5min, and PBS for 5min;
(8) DAPI staining was added for 15min, washed 3 times with pbs for 5min each, observed under a fluorescent microscope and photographed.
The experimental results are shown in fig. 1, and it can be seen that the number of the dyeing cells of the stephania polysaccharide with the concentration of 200mg/ml is significantly more than that of the stephania polysaccharide with the concentration of 0mg/ml, and the results further prove that the stephania polysaccharide can promote the proliferation of skeletal muscle cells of pigs.
Example 5
(1) Inoculating porcine skeletal muscle satellite cells into a 6-hole plate, culturing overnight, and replacing the porcine skeletal muscle satellite cells with a differentiation medium added with 0mg/ml stephania cepharantha polysaccharide, 100mg/ml stephania cepharantha polysaccharide and 200mg/ml stephania cepharantha polysaccharide;
(2) After 72h incubation, the medium was discarded, cells were washed with 1ml of PBS, 100 μl of cells were scraped off using a cell scraper, and transferred to an EP tube using a pipette;
(3) Cracking on ice for 30min, placing in a centrifuge, centrifuging at 4deg.C, 12000rpm/min, centrifuging for 15min;
(4) After centrifugation, carefully sucking the supernatant to obtain a protein sample;
(5) Protein concentration was measured using BCA method and protein sample concentration was adjusted to 2 μg/μl.
(6) Preparing 12% of separation gel and 5% of concentrated gel, and adding 10 μl of protein sample and protein Marker into each well;
(7) Concentrating gel at constant pressure of 90V, separating gel at constant pressure of 120V, and stopping electrophoresis when bromophenol blue runs to the bottom of gel;
(8) Carefully taking out the gel, mounting a transfer clamp according to the modes of the foam cushion, the three layers of filter paper, the PAGE gel, the PVDF film, the three layers of filter paper and the foam cushion, placing the transfer clamp into an electrotransfer groove, and electrically transferring the transfer clamp for 1.5 hours at room temperature;
(9) After the electric conversion is finished, the PVDF film is taken out and put into 5% skimmed milk powder, and the shaking table is closed for 1h at room temperature.
(10) After the closure was completed, the membrane was washed 3 times with TBST for 10 minutes each;
(11) Cutting PVDF membrane according to the size of the corresponding protein band, incubating MyHC, myoG and beta-actin primary antibodies at 4 ℃ for overnight;
(12) After the primary antibody incubation is finished, the membrane is washed 3 times by using TBST for 10 minutes each time, the corresponding 2 antibodies are incubated, and the incubation is carried out for 1h at room temperature;
(13) After the completion of the secondary antibody incubation, the membrane was washed 3 times with TBST, and development exposure was performed.
As a result, as shown in FIG. 2, it was found that the protein expression levels of MyHC and MyoG increased significantly with increasing concentration of the polysaccharide from Euphorbia lathyris.
Claims (3)
1. The application of the euphorbia lathyris polysaccharide in preparing the porcine skeletal muscle satellite cell differentiation promoting agent is characterized in that the preparation method of the euphorbia lathyris polysaccharide is as follows:
(1) Cleaning semen Euphorbiae, oven drying, and pulverizing into powder to obtain semen Euphorbiae powder;
(2) Adding petroleum ether-methanol mixed solution with the volume ratio of 2:1 in 20 times, degreasing for 5 hours to obtain degreased stephania japonica mixed solution;
(3) Filtering the defatted stephania japonica mixed solution to obtain stephania japonica filter residues;
(4) Adding 25 times of water into the euphorbia lathyris filter residue, and leaching for 5 hours at 85 ℃ to obtain euphorbia lathyris filter liquor;
(5) Filtering with ultrafiltration membrane with molecular weight cutoff of 10000 to remove impurities, and concentrating the semen Euphorbiae filtrate under reduced pressure to 1/3 of the original volume to obtain semen Euphorbiae Pulveratum Jin Zicu polysaccharide solution;
(6) Adding 4 times of 95% ethanol, precipitating with ethanol for 24h, centrifuging at 8000rpm for 10min, and removing supernatant to obtain thousands of Jin Zicu polysaccharides;
(7) Washing the Qian Jin Zicu polysaccharide with absolute ethanol and acetone, removing protein by sevage method, and freeze drying to obtain Qian jin Zi polysaccharide.
2. The application of the euphorbia lathyris polysaccharide in preparing a protein expression promoter of MyHC and MyoG in porcine skeletal muscle satellite cells is characterized in that the preparation method of the euphorbia lathyris polysaccharide is as follows:
(1) Cleaning semen Euphorbiae, oven drying, and pulverizing into powder to obtain semen Euphorbiae powder;
(2) Adding petroleum ether-methanol mixed solution with the volume ratio of 2:1 in 20 times, degreasing for 5 hours to obtain degreased stephania japonica mixed solution;
(3) Filtering the defatted stephania japonica mixed solution to obtain stephania japonica filter residues;
(4) Adding 25 times of water into the euphorbia lathyris filter residue, and leaching for 5 hours at 85 ℃ to obtain euphorbia lathyris filter liquor;
(5) Filtering with ultrafiltration membrane with molecular weight cutoff of 10000 to remove impurities, and concentrating the semen Euphorbiae filtrate under reduced pressure to 1/3 of the original volume to obtain semen Euphorbiae Pulveratum Jin Zicu polysaccharide solution;
(6) Adding 4 times of 95% ethanol, precipitating with ethanol for 24h, centrifuging at 8000rpm for 10min, and removing supernatant to obtain thousands of Jin Zicu polysaccharides;
(7) Washing the Qian Jin Zicu polysaccharide with absolute ethanol and acetone, removing protein by sevage method, and freeze drying to obtain Qian jin Zi polysaccharide.
3. An accelerator for promoting the differentiation of porcine skeletal muscle satellite cells, which is characterized by comprising stephania sinica diels polysaccharide and a pharmaceutically acceptable carrier;
the preparation method of the stephania polysaccharide comprises the following steps:
(1) Cleaning semen Euphorbiae, oven drying, and pulverizing into powder to obtain semen Euphorbiae powder;
(2) Adding petroleum ether-methanol mixed solution with the volume ratio of 2:1 in 20 times, degreasing for 5 hours to obtain degreased stephania japonica mixed solution;
(3) Filtering the defatted stephania japonica mixed solution to obtain stephania japonica filter residues;
(4) Adding 25 times of water into the euphorbia lathyris filter residue, and leaching for 5 hours at 85 ℃ to obtain euphorbia lathyris filter liquor;
(5) Filtering with ultrafiltration membrane with molecular weight cutoff of 10000 to remove impurities, and concentrating the semen Euphorbiae filtrate under reduced pressure to 1/3 of the original volume to obtain semen Euphorbiae Pulveratum Jin Zicu polysaccharide solution;
(6) Adding 4 times of 95% ethanol, precipitating with ethanol for 24h, centrifuging at 8000rpm for 10min, and removing supernatant to obtain thousands of Jin Zicu polysaccharides;
(7) Washing the Qian Jin Zicu polysaccharide with absolute ethanol and acetone, removing protein by sevage method, and freeze drying to obtain Qian jin Zi polysaccharide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210804681.1A CN115039841B (en) | 2020-09-02 | 2020-09-02 | Promoter for promoting differentiation of porcine skeletal muscle satellite cells |
Applications Claiming Priority (2)
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| CN111903860A (en) | 2020-11-10 |
| CN115039841A (en) | 2022-09-13 |
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