Amino acid culture medium for promoting synthesis of casein of dairy cow mammary gland cells and preparation method thereof

Abstract

The invention relates to the technical field of cells, and particularly discloses an amino acid culture medium for promoting synthesis of casein of dairy cow mammary cells and a preparation method. In the culture medium, the addition amount of lysine is 0.74 mmol / L, the addition amount of threonine is 0.22 mmol / L, and the addition amount of phenylalanine is 0.28 mmol / L. Tests prove that the culture medium under the condition of the application can obviously improve the relative proliferation rate and casein expression quantity of dairy cow mammary gland cells compared with a culture medium under 4 times of plasma concentration (optimal amino acid supply mode) of dairy cow. The method for promoting casein synthesis comprises the following steps: culturing cow mammary epithelial cells to P3 generation; when the P3 generation cells grow to 75-85% of the adherent area in a 75 cm < 3 > culture flask, transferring the P3 generation cells into a culture medium without lysine, threonine and phenylalanine, and starving for 16 hours; when the cells are hungry, transferring the cells into the culture medium prepared by the invention to be continuously cultured for 24 hours; and after culture is finished, measuring the relative proliferation rate and casein expression quantity of the cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cells, and relates to an amino acid culture medium for promoting casein synthesis of dairy cow mammary gland cells and a preparation method. BACKGROUND

[0002] Mammary gland cells play a crucial role in the milk synthesis process of dairy cow mammary glands, and casein, as one of the main proteins in milk, has high nutritional value and wide application prospects. At present, the culture of dairy cow mammary gland cells has become an important tool for studying the synthesis mechanism of casein.

[0003] Lysine, threonine and phenylalanine are essential amino acids for dairy cows, and their concentrations have a significant impact on the growth and protein synthesis of mammary gland cells. Among them, lysine, as the first limiting amino acid in the dairy cow body, directly participates in the formation of casein structure; threonine is not only an important component of casein, but also can activate the intracellular protein synthesis related signal pathway and promote protein translation; phenylalanine, as an aromatic amino acid, participates in the modification and folding process of casein, and plays a key role in maintaining the function and stability of casein. However, the existing culture medium still has deficiencies in the optimization of amino acid concentration. Most culture media usually rely on fixed amino acid concentrations, and fail to fully mobilize the potential of casein synthesis in mammary gland cells. Therefore, the development of a culture medium combined with the addition of lysine, threonine and phenylalanine can promote the synthesis of casein through the combined action of the three amino acids, thereby providing a new idea for improving the synthesis of dairy cow casein. SUMMARY

[0004] To achieve the above-mentioned purpose, the following technical solutions are adopted in the application: The application provides a culture medium for promoting casein synthesis of dairy cow mammary gland cells, which comprises DMEM / F12 culture medium, fetal bovine serum, double antibiotics, prolactin, hydrocortisone, insulin transferrin factor, epidermal growth factor, glutamine, lysine, threonine and phenylalanine. The DMEM / F12 culture medium is a commonly used culture medium for dairy cow mammary gland cells. The fetal bovine serum contains rich nutritional factors and is mainly used for in vitro culture of animal cells. The double antibiotics are penicillin and streptomycin. The prolactin, hydrocortisone, insulin transferrin factor, epidermal growth factor, glutamine, lysine, threonine and phenylalanine are growth factors required in the growth process of dairy cow mammary gland cells.

[0005] In the present application, the culture medium for promoting casein synthesis of cow mammary gland cells comprises 84.4% DMEM / F12 culture medium, 10% fetal bovine serum, 1.5% double antibody, 1% hydrocortisone, 1% insulin transferrin factor, 1% epidermal growth factor, 0.6% prolactin, and 0.5% glutamine in terms of mass fraction. In addition, the concentration of lysine ranges from 0.18 mmol / L to 2.94 mmol / L, the concentration of threonine ranges from 0.11 mmol / L to 0.88 mmol / L, and the concentration of phenylalanine ranges from 0.07 mmol / L to 0.56 mmol / L. More preferably, the concentrations of lysine, threonine and phenylalanine in the culture medium are 0.74 mmol / L, 0.22 mmol / L and 0.28 mmol / L, respectively.

[0006] In addition, the present application also provides a method for promoting casein synthesis of cow mammary gland cells, comprising: S01: culturing cow mammary gland cells in the above-mentioned culture medium to P3 generation; S02: when the P3 generation cow mammary gland cells grow to 75 cm 3 75-85% of the adherent area in the culture bottle, the cells are transferred to the above-mentioned culture medium without lysine, threonine and phenylalanine for 16 h of starvation; S03: after the cow mammary gland cells are starved, the cells are transferred to the above-mentioned culture medium for further culturing for 24 h; S04: after the culturing is completed, the relative proliferation rate of the cells ((test group OD value - control group OD value) / control group OD value x 100%) and the casein expression amount are measured.

[0007] The present application has the following beneficial effects: In the present application, after the cow mammary gland cells are normally cultured to P3 generation, the addition of 0.74 mmol / L lysine, 0.22 mmol / L threonine and 0.28 mmol / L phenylalanine in the culture medium can promote the proliferation of cow mammary gland cells and the expression amount of casein. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 Effect of different concentrations of lysine on the relative proliferation rate of cow mammary gland cells; Figure 2 Effect of different concentrations of lysine on the casein expression amount of cow mammary gland cells; Figure 3 Effect of different concentrations of threonine on the relative proliferation rate of cow mammary gland cells; Figure 4 Effect of different concentrations of threonine on the casein expression amount of cow mammary gland cells; Figure 5Effect of different concentrations of phenylalanine on the relative proliferation rate of dairy cow mammary gland cells Figure 6 Effect of different concentrations of phenylalanine on the expression of casein in dairy cow mammary gland cells Figure 7 Effect of combined addition of lysine, threonine and phenylalanine on the relative proliferation rate of dairy cow mammary gland cells Figure 8 Effect of combined addition of lysine, threonine and phenylalanine on the expression of casein in dairy cow mammary gland cells DETAILED DESCRIPTION

[0009] The technical solutions of the present application are further explained and described below through specific experimental design.

[0010] The present application includes two parts of experiments:

[0011] Single factor complete random experimental design was adopted, and a customized DMEM / F12 medium without 9 essential amino acids was used as the basis to prepare a culture medium containing different concentrations of lysine (0.18, 0.37, 0.74, 1.47 and 2.94 mmol / L), threonine (0.11, 0.22, 0.44 and 0.88 mmol / L) and phenylalanine (0.07, 0.14, 0.28 and 0.56 mmol / L), and the addition levels of the other 6 essential amino acids were 4 times the amino acid concentration in cow plasma (referring to the paper published by Sun et al. in 2025). There were 6 replicates in each treatment group. After the culture was completed, the cells were collected for analysis to determine the relative proliferation rate and casein expression.

[0012] It can be known that the relative proliferation rate of dairy cow mammary gland cells in the 1.47 mmol / L lysine group was significantly higher than that in the other groups ( Figure 1 <0.05). P

[0013] It can be known that the expression of casein in the 0.74 mmol / L lysine group was significantly higher than that in the other groups ( Figure 2 <0.05); and the expression of casein in the 1.47 mmol / L lysine group was significantly higher than that in the 0.18 mmol / L and 2.94 mmol / L groups ( P <0.05). P

[0014] It can be known that the relative proliferation rate of dairy cow mammary gland cells in the 0.11 mmol / L threonine group was significantly higher than that in the other groups ( Figure 3 <0.05). P

[0015] Figure 4 ​​​​It can be seen that the casein expression in the 0.11 mmol / L threonine group was significantly higher than that in the 0.44 mmol / L and 0.88 mmol / L groups ( P < 0.05); there was a trend higher than the 0.22 mmol / L group, but the difference was not significant ( P > 0.05).

[0016] Depend on Figure 5 It can be seen that when the concentration of phenylalanine increased from 0.07 mmol / L to 0.56 mmol / L, it had no significant effect on the proliferation rate of bovine mammary cells ( P > 0.05).

[0017] Depend on Figure 6 It can be seen that the expression of casein in the 0.14 mmol / L phenylalanine group was significantly higher than that in the 0.07 mmol / L and 0.56 mmol / L groups ( P < 0.05), and there was no significant difference between the 0.28 mmol / L group ( P > 0.05).

[0018] 7. Conclusion: Lysine (0.74 mmol / L and 1.47 mmol / L), threonine (0.11 mmol / L and 0.22 mmol / L) and phenylalanine (0.14 mmol / L and 0.28 mmol / L) are the optimal concentrations for promoting casein synthesis in bovine mammary cells.

[0019] Based on the results of the single-factor experiment, a three-factor, two-level (2³) orthogonal design was used, with four treatment groups (see Table 1). The control group (CON) received 4 times the plasma essential amino acid concentration of dairy cows (referring to the paper published by Sun et al. in 2025). Each treatment was replicated six times. After culture, cells were collected and analyzed to determine relative cell proliferation rate and casein expression.

[0020] Table 1 Essential amino acid supplementation levels in each treatment group of orthogonal experimental design

[0021] Depend on Figure 7 It can be seen that different combinations of lysine, threonine and phenylalanine have no significant effect on cell viability ( P >0.05).

[0022] Depend on Figure 8 It can be seen that the expression of casein in experimental groups Ⅰ, Ⅱ and Ⅳ was significantly higher than that in the control group and experimental group Ⅲ ( P <0.05), among which the expression of casein in test group II was significantly higher than that in test groups I and IV ( P < 0.05).

[0023] 3、Conclusion: the combination of 0.74 mmol / L lysine, 0.22 mmol / L threonine and 0.28 mmol / L phenylalanine (test group II) is a suitable ratio for promoting casein synthesis.

[0024] The above merely describes the preferred embodiments of the present application, and it should be noted that those of ordinary skill in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. An amino acid culture medium for promoting casein synthesis in bovine mammary cells, characterized in that: Including DMEM / F12 culture medium, fetal bovine serum, double antibody, hydrocortisone, insulin-transferrin factor, epidermal growth factor, prolactin, glutamine, lysine, threonine, and phenylalanine.

2. The culture medium for promoting casein synthesis in bovine mammary cells according to claim 1, characterized in that The culture medium includes, by mass fraction, 84.4% DMEM / F12 culture medium, 10% fetal bovine serum, 1.5% double-antibody, 1% hydrocortisone, 1% insulin-transferrin factor, 1% epidermal growth factor, 0.6% prolactin, and 0.5% glutamine.

3. The culture medium for promoting casein synthesis in bovine mammary cells according to claim 1, characterized in that The dual antibody is penicillin and streptomycin.

4. The culture medium for promoting casein synthesis in bovine mammary cells according to claim 1, characterized in that The lysine concentration in the culture medium ranged from 0.18 mmol / L to 2.94 mmol / L, the threonine concentration ranged from 0.11 mmol / L to 0.88 mmol / L, and the phenylalanine concentration ranged from 0.07 mmol / L to 0.56 mmol / L.

5. A method for promoting casein synthesis in bovine mammary cells, characterized in that: include: S101: Cultivating bovine mammary cells in any one of the culture media of claims 1-4 to the P3 generation; S102: When the P3 generation bovine mammary cells grow to 75-85% of the adherent area in a 75 cm 3 culture flask, transfer them to any one of the culture media of claims 1-4 without lysine, threonine and phenylalanine and starve them for 16 hours; S103: After the cow mammary cells are starved, transferring them to any one of the culture media according to claims 1-4 and continuing to culture for 24 hours; S104: After the culture is completed, the relative proliferation rate and casein expression level of the cow mammary gland cells are measured.

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