Application of myricetin-3-o-galactoside in induction of casein expression in mammary epithelial cells

Abstract

The invention provides application of myricetin-3-o-galactoside in induction of casein expression in mammary epithelial cells, and belongs to the technical field of cell culture. The invention provides an application of myricetin-3-o-galactoside in at least one of the following aspects: promoting cell growth, inducing casein expression in cells and constructing a casein expression dominant cell model. The myricetin-3-o-galactoside can be used for inducing the expression of casein in cells and promoting the growth of the cells. The embodiment of the invention shows that after myricetin-3-o-galactoside is added into the culture medium, the casein expression quantity in mammary epithelial cells is improved by 5.1 times, and the cell growth rate is also obviously improved. In addition, the method is high in repeatability and stable in induction effect, and a new path is provided for improving the yield and quality of dairy products.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and particularly relates to an application of myricetin-3-O-galactoside in inducing casein expression in mammary epithelial cells. Background Art

[0002] Casein content is a key factor in assessing milk quality. Higher casein content generally indicates higher nutritional value and greater consumer appeal. Therefore, increasing casein content has long been a goal in dairy processing and quality control.

[0003] Mammary epithelial cells possess the specific function of synthesizing and secreting milk. They can synthesize and secrete milk proteins through in vitro induction, making them an ideal model for studying the mechanisms regulating lactation. Existing methods for inducing mammary epithelial cells to secrete casein are inefficient and have poor reproducibility. For example, studies have shown that casein expression increased 2.4-fold after induction with DMEM / F12 culture medium, insulin, hydrocortisone, and valine. However, after 72 hours of induction with DMEM / F12 culture medium, insulin, hydrocortisone, and phenylalanine, casein expression only increased 1.7-fold, indicating low induction efficiency. In the prior art (Lee, HY, Heo, YT, Lee, SE, Hwang, KC, Lee, HG, Choi, SH, & Kim, NH (2013). Short communication: retinoic acid plus prolactin to synergistically increase specific casein gene expression in MAC-T cells. J Dairy Sci, 96(6), 3835-3839. doi: 10.3168 / jds.2012-5945), retinoic acid (RA) was added to the basal culture medium to synergize with hydrocortisone (HC), insulin (INS), and prolactin (PRL). The expression of casein was increased compared with the control group, but the results of repeated experiments were quite different. The existing induction methods have low induction efficiency and irreproducible experimental results, which seriously restrict the development of casein induction technology. Summary of the Invention

[0004] In view of this, the present invention provides a new application of myricetin-3-o-galactoside, which can effectively induce the expression of casein with high induction efficiency and stable effect.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides an application of myricetin-3-o-galactoside in at least one of the following: inducing casein expression in cells, constructing a casein-expressing dominant cell model, and promoting cell growth.

[0007] Preferably, the cells comprise mammary epithelial cells.

[0008] The invention provides a cell induction culture medium, comprising a sugar-free DMEM / F12 culture medium containing the following components: 2-10 μg / mL of myricetin-3-o-galactoside, 7%-11% by volume of fetal bovine serum, 4-6 μg / mL of bovine insulin, 0.8-1.2 μg / mL of hydrocortisone, 4-6 μg / mL of prolactin, and 0.8-1.2 μM of retinoic acid.

[0009] Preferably, the sugar-free DMEM / F12 culture medium comprises the following components: myricetin-3-o-galactoside 3-8 μg / mL, fetal bovine serum 8%-10% by volume, bovine insulin 5 μg / mL, hydrocortisone 1 μg / mL, prolactin 5 μg / mL and retinoic acid 1 μM.

[0010] The present invention provides an application of the cell induction culture medium in at least one of the following: promoting cell growth, inducing casein expression in cells, and constructing a casein-expressing dominant cell model.

[0011] Preferably, the cells comprise mammary epithelial cells.

[0012] The present invention provides a method for promoting casein expression in mammary epithelial cells, which is characterized in that the mammary epithelial cells are induced and cultured using the cell induction culture medium.

[0013] Preferably, the induction culture time is 36 to 60 hours.

[0014] Preferably, the mammary epithelial cells include mammary epithelial cells that have been starved;

[0015] The starvation treatment lasts for 14 to 18 hours.

[0016] Preferably, the mammary epithelial cells include primate mammary epithelial cells or bovine mammary epithelial cells.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The present invention provides an application of myricetin-3-o-galactoside in at least one of the following: inducing casein expression in cells, constructing a casein expression-dominant cell model, and promoting cell growth. In the present invention, the myricetin-3-o-galactoside can efficiently induce casein expression in cells and promote cell growth. The embodiments of the present invention show that the induction culture medium containing myricetin-3-o-galactoside increases the casein expression in mammary epithelial cells by 5.1 times compared to the induction culture medium not containing myricetin-3-o-galactoside, and the growth rate is significantly improved. Moreover, the induction efficiency is high and stable after repeated inductions. It can be seen that the present invention provides a reliable and efficient casein induction scheme.

[0019] The present invention provides a cell induction culture medium comprising a sugar-free DMEM / F12 culture medium containing the following components: 2-10 μg / mL myricetin-3-o-galactoside, 7%-11% by volume fetal bovine serum, 4-6 μg / mL bovine insulin, 0.8-1.2 μg / mL hydrocortisone, 4-6 μg / mL prolactin, and 0.8-1.2 μM retinoic acid. In the present invention, the sugar-free DMEM / F12 and fetal bovine serum provide nutrients to cells and promote casein expression in mammary epithelial cells. The insulin, hydrocortisone, prolactin, and retinoic acid promote cell growth, induce mammary epithelial cells to enter a lactation state, increase CSN2 gene expression levels in the cells, and allow casein expression. The myricetin-3-o-galactoside regulates cellular energy metabolism and hormone signaling pathways, promotes casein synthesis, and significantly increases casein expression. The present invention demonstrates that the addition of myricetin-3-O-galactoside to a sugar-free DMEM / F12 culture medium containing fetal bovine serum, insulin, hydrocortisone, prolactin, and retinoic acid effectively promotes cell growth and increases casein expression in bovine mammary epithelial cells. The expression of casein in the control group without myricetin-3-O-galactoside was increased by 5.1 times. This demonstrates that the present invention provides an efficient culture medium for inducing casein expression.

[0020] The present invention provides a method for promoting casein expression in mammary epithelial cells, wherein the mammary epithelial cells are induced and cultured using the cell induction culture medium. The method of the present invention can effectively promote mammary epithelial cells to enter a lactation state, increase the expression of casein, and promote the growth of lactating mammary epithelial cells. Transcriptome detection results show that the method can induce changes in the expression of AKAP7, TXNIP, histone H4, etc., and increase the synthesis of milk proteins and increase the expression of casein by regulating hormone signaling pathways, energy metabolism and gene expression during lactation. In addition, the method of the present invention has high biological repeatability, and multiple experiments have shown that the induction efficiency is high and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the process for inducing casein secretion from mammary epithelial cells in vitro.

[0022] Figure 2 This is the result of detecting casein expression after MACT-1 induction;

[0023] Figure 3 This is the result of detecting casein expression after MACT-2 induction;

[0024] Figure 4 This is the result of detecting casein expression after MACT-3 induction;

[0025] Figure 5 The figure shows the results of the casein expression test after induction of MACT-4(1) and MACT-4(2);

[0026] Figure 6 This is the result of detecting casein expression after MACT-5 induction;

[0027] Figure 7 This is the morphology of MAC-T cells after MACT-5 induction;

[0028] Figure 8 The results of differential gene analysis for transcriptome detection;

[0029] Figure 9 This is the result diagram of gene enrichment analysis;

[0030] Figure 10 This is the concentration optimization result of myricetin-3-O-galactoside. DETAILED DESCRIPTION

[0031] The present invention provides an application of myricetin-3-o-galactoside in at least one of the following: inducing casein expression in cells, constructing a casein-expressing dominant cell model, and promoting cell growth.

[0032] In the present invention, the cells preferably include mammary epithelial cells. These mammary epithelial cells preferably include primate mammary epithelial cells or bovine mammary epithelial cells, and more preferably include human mammary epithelial cells or bovine mammary epithelial cells. Mammary epithelial cells have the specific function of synthesizing and secreting milk and are widely used in production practices, providing powerful cell model support for the development of mammary bioreactors and other aspects.

[0033] In the present invention, by screening the differential metabolites of bovine mammary epithelial cells in different induction culture media, the results show that the content of myricetin-3-o-galactoside increases in a low-sugar environment, and mammary epithelial cells are cultured with an induction culture medium containing the myricetin-3-o-galactoside, which can efficiently induce casein expression in the cells and promote cell growth. Mammary epithelial cells are placed in an induction culture medium containing myricetin-3-o-galactoside and cultured to obtain a casein expression advantage cell model. The embodiments of the present invention show that the induction culture medium containing myricetin-3-o-galactoside increases the casein expression in mammary epithelial cells by 5.1 times compared to the induction culture medium without myricetin-3-o-galactoside, and the induction efficiency is high and stable after repeated induction.

[0034] The invention provides a cell induction culture medium, which comprises a sugar-free DMEM / F12 culture medium containing the following components: 2-10 μg / mL of myricetin-3-o-galactoside, 7%-11% by volume of fetal bovine serum, 4-6 μg / mL of bovine insulin, 0.8-1.2 μg / mL of hydrocortisone, 4-6 μg / mL of prolactin, and 0.8-1.2 μM of retinoic acid.

[0035] In the present invention, the cell induction medium preferably includes a sugar-free DMEM / F12 medium containing the following components: myricetin-3-o-galactoside 3-8 μg / mL, fetal bovine serum 8% to 10% by volume, bovine insulin 5 μg / mL, hydrocortisone 1 μg / mL, prolactin 5 μg / mL and retinoic acid 1 μM.

[0036] In the present invention, described sugar-free DMEM / F12 culture medium is preferably obtained by mixing equal volumes of sugar-free DMEM culture medium and F12 culture medium.The volume percentage of described foetal calf serum is most preferably 9%.The sugar-free DMEM / F12 culture medium containing foetal calf serum can promote the expression of casein in mammary epithelial cells.In embodiments of the present invention, compared the impact of the DMEM / F12 culture medium of the different sugar concentrations adding foetal calf serum on bovine mammary epithelial cells, result shows, sugar-free DMEM / F12 culture medium improves 2.6 times compared to control group (RPIM1640 culture medium) bovine mammary epithelial cells, and high sugar DMEM / F12 culture medium only improves 1.6 times compared to control group.As seen, the sugar-free DMEM / F12 culture medium induction efficiency containing foetal calf serum is better, is conducive to improving the casein expression in bovine mammary epithelial cells.

[0037] In the present invention, the sugar-free DMEM / F12 medium preferably further comprises antibiotics, preferably a penicillin-streptomycin mixture (double antibiotic), the volume percentage of the penicillin-streptomycin mixture in the sugar-free DMEM / F12 medium preferably being 1%. The antibiotics can prevent bacterial contamination of cells and improve induction efficiency and stability.

[0038] In the present invention, the insulin, hydrocortisone, prolactin, and retinoic acid can promote cell growth, induce mammary epithelial cells to enter a lactation state, significantly increase the expression level of the CSN2 gene in the cells, and express casein. The present invention demonstrates that the cell culture medium increases casein expression in bovine mammary epithelial cells by 4.2 times compared to a control group (RPIM1640 culture medium), effectively promoting cell growth and increasing casein expression in bovine mammary epithelial cells.

[0039] In the present invention, the concentration of the myricetin-3-o-galactoside is further preferably 4 to 6 μg / mL, and most preferably 5 μg / mL. Adding the myricetin-3-o-galactoside to the sugar-free DMEM / F12 culture medium containing fetal bovine serum, insulin, hydrocortisone, prolactin and retinoic acid can further promote the growth of mammary epithelial cells and efficiently induce the expression of casein. In the examples of the present invention, the experimental results show that compared with the control group to which myricetin-3-o-galactoside is not added, the culture medium containing myricetin-3-o-galactoside increases the expression of casein in mammary epithelial cells by 5.1 times, and at the same time, the growth rate of the cells is increased, thereby promoting cell growth. It can be seen that the present invention provides an efficient casein induction culture medium.

[0040] In view of the characteristics of the cell induction culture medium that promotes cell growth and induces casein expression, the present invention provides a use of the cell induction culture medium in at least one of the following: inducing casein expression in cells, constructing a casein-expressing dominant cell model, and promoting cell growth.

[0041] In the present invention, the cells preferably include mammary epithelial cells. Cultivating mammary epithelial cells with the cell induction culture medium can efficiently induce casein expression in the cells and improve the growth of mammary epithelial cells. Adopting the cell induction culture medium to induce and culture mammary epithelial cells can obtain a cell model with superior casein expression. The results of the embodiment of the present invention show that the cell induction culture medium can increase the expression of casein in bovine mammary epithelial cells, increase the growth rate of mammary epithelial cells, and promote the growth of mammary epithelial cells.

[0042] The invention provides a method for promoting casein expression in mammary epithelial cells, and adopts the cell induction culture medium to carry out induction culture on the mammary epithelial cells.

[0043] In the present invention, the mammary epithelial cells preferably include mammary epithelial cells that have undergone starvation treatment. The starvation-treated mammary epithelial cells are preferably obtained by transferring subcultured mammary epithelial cells to a starvation treatment medium when the cells have grown to 40%. The subculture medium preferably includes DMEM medium or 1640 medium containing fetal bovine serum and antibiotics, more preferably RPIM1640 medium containing fetal bovine serum and antibiotics. In an embodiment of the present invention, RPIM1640 medium is purchased from Gibco, catalog number 11875119. The volume percentage of fetal bovine serum is preferably 8% to 12%, more preferably 9% to 11%, and most preferably 10%. The antibiotics preferably include a mixture of penicillin and streptomycin. The total mass percentage of the penicillin-streptomycin mixture in the sugar-free DMEM / F12 medium is 1%. The subculture temperature is preferably 37-38°C, more preferably 37.5°C. Subculture can enhance cell viability and facilitate the cells' lactation after subsequent starvation treatment. The culture medium for the starvation treatment preferably includes DMEM culture medium or 1640 culture medium containing antibiotics, more preferably includes RPIM1640 culture medium. The antibiotics preferably include penicillin and streptomycin, more preferably include a 1.1% penicillin-streptomycin mixture (double antibody). The temperature of the starvation treatment is preferably the same as the temperature of the subculture, which will not be repeated here. The time of the starvation treatment is preferably 14 to 18 hours, more preferably 15 to 17 hours, and more preferably 16 hours. Induction culture after the starvation treatment is beneficial to inducing cells to enter the lactation state, increasing the expression of casein, and promoting the growth of mammary epithelial cells in the lactation state.

[0044] In the present invention, the temperature of the induction culture is preferably the same as the temperature of the subculture, which will not be repeated here. During the induction culture process, when the culture medium is replaced, the culture medium is preheated. The preheating temperature is preferably 37-38°C, and more preferably 37.5°C. Preheating of the culture medium can reduce the stimulation to mammary epithelial cells, promote the secretion of casein by mammary epithelial cells, and promote the growth of lactating mammary epithelial cells. In the embodiment of the present invention, the mammary epithelial cells are cultured in an incubator, and the incubator is set to a temperature of 37.5°C. The time of the induction culture is preferably 36-60h, more preferably 40-56h, and most preferably 48h. The induction time of the present invention has a high efficiency for casein induction in mammary epithelial cells.

[0045] In the present invention, the mammary epithelial cells preferably include primate mammary epithelial cells or bovine mammary epithelial cells, and more preferably include human mammary epithelial cells or bovine mammary epithelial cells.

[0046] The method of the present invention can effectively induce mammary epithelial cells to enter a lactation state, increase the expression of casein in mammary epithelial cells, and promote the growth of lactating mammary epithelial cells. The examples of the present invention show that the method can increase the expression of casein at the RNA level. At the same time, the transcriptome detection results show that the method regulates hormone signaling pathways, energy metabolism and gene expression during lactation, thereby increasing the synthesis of milk protein and increasing the expression of casein. In addition, the method of the present invention has high biological reproducibility, and multiple experiments have shown that the induction efficiency is stable.

[0047] In order to further illustrate the present invention, the solutions provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Sources:

[0049] Bovine mammary epithelial cells (MAC-T) were derived from the same source as the bovine mammary epithelial cells (BMECs) used in the prior art (Cheng Xiaoou. Mechanism of Staphylococcus aureus invading bovine mammary epithelial cells and inducing heterophagy [D]. Inner Mongolia University, 2021. DOI: 10.27224 / d.cnki.gnmdu.2021.000032.) and were stored in Laboratory 432, School of Life Sciences, Inner Mongolia University. Fetal bovine serum was purchased from BI, bovine insulin and hydrocortisone from Solebao, prolactin from PROSPEC, retinoic acid from Sigma-Aldrich, myricetin-3-o-galactoside from BioBiopha, and penicillin-streptomycin mixture (double antibody) (PS) from Gibco.

[0050] RPIM1640 medium, DMEM / F12 medium, sugar-free DMEM medium, and F12 medium were all purchased from Gibco. The F12 product number is 31765035, and the DMEM product number is A1443001.

[0051] Example 1

[0052] The method for inducing bovine mammary epithelial cells to secrete casein in vitro comprises the following steps:

[0053] 1. Culture and passage of bovine mammary epithelial cells (MAC-T):

[0054] The bovine mammary epithelial cells in the incubator were taken out, the culture medium in the wells was aspirated, 1 mL of DPBS buffer was slowly added to the wells to adhere to the wall, the culture plate was gently shaken several times, the wash solution in the wells was aspirated to remove dead cells and impurities, 500 μL of preheated trypsin (TRYPSIN) was immediately added to the wells, and the cells were placed in the incubator for 3 minutes. If the cells were basically no longer adhered to the wall as observed under a microscope, 1500 μL of preheated RPIM1640 culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture (double antibody) (PS) was added, and the cell suspension was repeatedly pipetted several times. The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged (1300 rpm, 3 min). The supernatant in the centrifuge tube was aspirated and 1 mL of RPIM1640 culture medium containing 9% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture (double antibody) (PS) was added to resuspend the cell pellet and then connected to a 6-well plate and placed in the incubator for culture. Observe the growth morphology of MAC-T cells daily and change the culture medium daily to ensure adequate nutrition for the cells. Excess cells can be frozen. Each 10 mL of 10% DMSO freezing medium contains: 1 mL of DMSO and 9 mL of FBS.

[0055] 2. Starvation Culture of MAC-T Cells

[0056] When the cells reach approximately 40% growth, remove the cells from the incubator and discard the culture medium in the wells. Slowly add 1 mL of DPBS buffer to the wells where they adhere to the wall. Gently shake the plate several times and discard the wash solution to remove dead cells and impurities. Immediately add starvation medium after discarding the DPBS and continue incubating in the incubator for 16 hours to obtain starvation cultured cells.

[0057] The starvation culture medium was RPIM1640 culture medium containing 1.1% penicillin-streptomycin mixture (double antibody) (PS).

[0058] 3. MAC-T Induction Culture

[0059] The starvation cultured cells were removed from the incubator, the culture medium in the wells was aspirated, and 1 mL of DPBS buffer was slowly added to the wells where they adhered to the wall. The culture plate was gently shaken several times, and the wash solution in the wells was aspirated to remove dead cells and impurities. The cells were then randomly divided into 7 groups for induction culture, which were designated as the control group, MACT-1, MACT-2, MACT-3, MACT-4(1), MACT-4(2), and MACT-5. The control group was added with 2 ml of pre-heated RPIM1640 culture medium and cultured in the incubator for 2 days. The remaining groups were added with 2 ml of different pre-heated induction culture medium and cultured in the incubator for 2 days. The composition of the induction culture medium for the remaining components is shown in Tables 1 to 6.

[0060] Table 1 MACT-1 induction culture medium composition

[0061]

[0062]

[0063] Table 2 MACT-1 induction culture medium composition

[0064] Element content F12 45mL PS 0.5mL Fetal bovine serum (FBS) 5％ Bovine insulin (INS) 5 μg / ml Hydrocortisone (HC) 1 μg / ml Prolactin (PRL) 5 μg / m Retinoic acid (RA) 1 μM

[0065] Table 3 MACT-1 induction culture medium composition

[0066] Element content Sugar-free DMEM 22.25ml F12 22.25mL PS 0.5mL Fetal bovine serum (FBS) 5％ Bovine insulin (INS) 5 μg / ml Hydrocortisone (HC) 1 μg / ml Prolactin (PRL) 5 μg / m Retinoic acid (RA) 1 μM

[0067] Table 4 MACT-4 (1) induction culture medium composition

[0068] Element content Sugar-free DMEM 22.25mL F12 22.25ml PS 0.5mL Fetal bovine serum (FBS) 5％

[0069] Table 5 MACT-4 (2) induction culture medium composition

[0070]

[0071]

[0072] Table 6 MACT-5 induction culture medium composition

[0073] Element content Sugar-free DMEM 22.25mL F12 22.25mL PS 0.5mL Fetal bovine serum (FBS) 4.5mL Bovine insulin (INS) 5 μg / mL Hydrocortisone (HC) 1 μg / mL Prolactin (PRL) 5 μg / mL Retinoic acid (RA) 1 μM Myricetin-3-o-galactoside 5 μg / mL

[0074] Example 2

[0075] 1. Detection of casein expression at the RNA level

[0076] After 2 days of induction culture of each group of MAC-T cells in Example 1, RNA of each group of MAC-T cells was extracted, and the expression level of casein was detected at the RNA level, as follows.

[0077] Gene expression detection at the RNA level: Add RNAiso Plus (500μL / well), let it stand for 5 minutes, and collect it in a 1.5mL sterile enzyme-free centrifuge tube; add 100μL chloroform (the added volume is 1 / 5 of RNAiso Plus) to the 1.5mL sterile enzyme-free centrifuge tube, shake vigorously for 15s to emulsify it, let it stand at room temperature for 5 minutes, and centrifuge at 4℃ (12000rpm, 15min); at this time, the mixture is divided into three layers. Carefully transfer 2 / 3 of the top layer of liquid to another new 1.5mL sterile enzyme-free centrifuge tube and record the volume (usually 200-250μL); add an equal volume of isopropanol to the above, carefully mix the liquid, let it stand at room temperature for 10 minutes, and centrifuge at 4℃ (12000rpm, 10min). At this time, a small flake white precipitate will appear at the bottom of the centrifuge tube; discard the supernatant and slowly add 1mL along the wall of the centrifuge tube. Wash the tube walls with 75% ethanol by gently inverting. Centrifuge at 4°C (12,000 rpm, 15 minutes). Discard the supernatant, aspirate the tube, and remove as much liquid as possible. Place in a fume hood for 5-8 minutes. Once the precipitate is no longer visible, add 20-25 μL of RNase-free water, dispersing the droplets evenly along the tube walls and bottom. Once the RNA is completely dissolved, aspirate and determine the RNA concentration. Any remaining RNA samples can be stored in a -80°C freezer. Prepare a 10 μL total reaction mixture in a PCR tube: 7 μL of RNase-free ddH2O, 1 μL of dNTP Mixture, 1 μL of Oligo dT Primer, and 1 μg of RNA template. Incubate the reaction mixture at 55°C for 5 minutes and then immediately cool on ice.

[0078] Prepare the following reaction mixture in a total volume of 20 μL: 10 μL of prepared RNA substrate, 5 μL of RNase-Free ddH2O, 4 μL of 5x PrimeScript Buffer, 1 μL of RNase Inhibitor, and 1 μL of PrimeScript RNase. After gentle mixing, incubate the reaction mixture at 37°C for 1 hour. Subsequently, incubate at 95°C for 5 minutes to inactivate the enzyme and immediately cool on ice or freeze at -80°C for long-term storage. Next, in an opaque PCR tube, prepare the following reaction mixture in a total volume of 20 μL, protected from light: 10 μL of SYBR Premix Ex Taq (purchased from Nanjing Novozymes Biotechnology Co., Ltd.), 2 μL of primers (1 μL each of forward and reverse primers), 2 μg of cDNA template, and 6 μL of RNase-Free ddH2O.

[0079] CSN2 forward primer: AGGAACAGCAGCAAACAG (SEQ ID NO: 1)

[0080] CSN2 reverse primer: TTTCCAGTCGCAGTCAAT (SEQ ID NO: 2)

[0081] GAPDH forward primer: ACGGCACAGTCAAGGCAGA (SEQ ID NO: 3)

[0082] GAPDH forward primer: GTGATGGCGTGGACAGTGG (SEQ ID NO: 4)

[0083] After correctly arranging the above reactants, place them in the PCR instrument. Set the following cycling parameters: initial denaturation at 95°C for 2 minutes, followed by 38 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 30 seconds. Finally, store at 4°C.

[0084] Test results are shown in Figures 2 to 7 . Figure 2 The results showed that the expression of casein in bovine mammary epithelial cells increased by 1.9 times after MACT-1 induction; Figure 3 The results showed that the expression of casein in bovine mammary epithelial cells increased 2-fold after MACT-2 induction; Figure 4 The results showed that the expression of casein in bovine mammary epithelial cells increased 4.2-fold after MACT-3 induction; Figure 5 The results showed that the expression of casein in bovine mammary epithelial cells increased by about 2.6 times after induction by MACT-4(1), and by 1.6 times after induction by MACT-4(2). Figure 5 It can be seen that the induction efficiency of casein is highest in a low-sugar environment (a mixture of sugar-free DMEM culture medium and F12 culture medium at a ratio of 1:1). Figure 6 The results showed that MACT-5 (with myricetin-3-o-galactoside) showed a significant upward trend in casein expression compared to MACT-3, with the expression level further increased by 5.1 times compared to MACT-3. All results of the present invention were biologically replicated, and multiple experiments proved that the induction efficiency was stable.

[0085] 2. Transcriptome detection for differential gene analysis

[0086] Transcriptome detection results ( Figure 8) showed that the differentially expressed genes mainly included AKAP7, TXNIP, histone H4, etc. AKAP7 plays a key role in the hormone signaling pathway during lactation, such as the prolactin signaling pathway. As a protein kinase A anchoring protein, AKAP7 can regulate the localization and activity of protein kinase A (PKA), and the PKA signaling pathway plays an important role in hormone signal transduction in many cells. TXNIP plays an important role in cellular energy metabolism. It can affect the uptake and utilization of glucose by cells by regulating the expression and function of glucose transporters GLUT1 and GLUT4. Histone H4 can change the structure and function of chromatin and affect gene expression through various modifications, such as acetylation, methylation, phosphorylation, etc. Figure 9 As can be seen in the figure, two genes are enriched in the positive regulation process of STAT protein synthesis. STAT5 can bind to the promoter region of milk protein genes such as β-casein, promoting the transcription of these genes and thus increasing milk protein synthesis.

[0087] Example 3

[0088] Method for inducing mammary epithelial cells to secrete casein in vitro

[0089] The concentration of myricetin-3-O-galactoside was explored to obtain the optimal induction conditions. The induction method for inducing mammary epithelial cells to secrete casein in vitro was the same as that of MACT-5 in Example 1, with the only difference being that the concentration of myricetin-3-O-galactoside was set to 5 μg / ml, 15 μg / ml or 10 μg / ml. Figure 10 ,Depend on Figure 10 It can be seen that the cell induction medium supplemented with 5 μg / ml or 10 μg / ml myricetin-3-O-galactoside has a better induction effect.

[0090] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of myricetin-3-o-galactoside in at least one of the following: inducing casein expression in cells, constructing a casein-expressing cell model, and promoting cell growth.

2. The application according to claim 1, characterized in that The cells include mammary epithelial cells.

3. A cell induction medium, characterized in that The sugar-free DMEM / F12 culture medium comprises the following components: myricetin-3-o-galactoside 2-10 μg / mL, fetal bovine serum volume percentage 7%-11%, bovine insulin 4-6 μg / mL, hydrocortisone 0.8-1.2 μg / mL, prolactin 4-6 μg / mL and retinoic acid 0.8-1.2 μM.

4. The cell induction medium according to claim 3, characterized in that The sugar-free DMEM / F12 culture medium comprises the following components: myricetin-3-o-galactoside 3-8 μg / mL, fetal bovine serum 8%-10% by volume, bovine insulin 5 μg / mL, hydrocortisone 1 μg / mL, prolactin 5 μg / mL and retinoic acid 1 μM.

5. Use of the cell induction medium according to claim 3 or 4 in at least one of the following: inducing casein expression in cells, constructing a casein-expressing dominant cell model, and promoting cell growth.

6. The application according to claim 5, characterized in that The cells include mammary epithelial cells.

7. A method for promoting casein expression in mammary epithelial cells, characterized in that: Mammary epithelial cells are induced and cultured using the cell induction culture medium according to claim 3 or 4.

8. The method according to claim 7, characterized in that: The induction culture time is 36 to 60 hours.

9. The method according to claim 7, characterized in that: The mammary epithelial cells include mammary epithelial cells treated with starvation; The starvation treatment lasts for 14 to 18 hours.

10. The method according to any one of claims 7 to 9, characterized in that: The mammary epithelial cells include primate mammary epithelial cells or bovine mammary epithelial cells.

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